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Alkaloid rich fraction of Datura alba Rumph. ex Nees leaves possesses antitumor and antimitotic activity
South African Journal of Botany 117 (2018) 282–287
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Alkaloid rich fraction of Datura alba Rumph. ex Nees leaves possesses antitumor and antimitotic activity Z. Saddiqe ⁎, U. Wahab, A. Maimoona, R. Raheel, M. Iram Department of Botany, Lahore College for Women University, Lahore, Pakistan
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Article history: Received 15 December 2017 Received in revised form 26 April 2018 Accepted 30 May 2018 Available online xxxx Edited by JJ Nair Keywords: Agrobacterium tumefaciens Allium cepa root tip Alkaloids Antimitotic Antitumor Datura alba
a b s t r a c t The antimitotic and antitumor activities of total alkaloids extracted from leaves of Datura alba Nees. were investigated using in vitro methods. The plant leaves were collected from local ground of Lahore College for Women University. The antitumor bioassay was performed using potato discs applied with plant alkaloid extract and activity was checked against Agrobacterium tumefaciens. The results demonstrated that the alkaloid rich fraction significantly repressed the tumor growth on potato discs at a concentration of 1000 ppm, giving 84.78% tumor inhibition after 21 days of incubation. The antimitotic bioassay was implemented using Allium cepa root tip bioassay. The findings suggested that the alkaloidal fraction also showed significant antimitotic effect on root tip cells of A. cepa. At 1000 ppm concentration, the alkaloidal extract revealed best antimitotic activity giving reduced (30 ± 0.577%) mitotic index after 72 h of treatment. The outcome of the present experiment, therefore, indicates that Datura alkaloids could be regarded as a potential source for the development of anti-tumor and antimitotic agents. The study could be extended further for drug development against cancers in humans. © 2018 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Human beings have been utilizing medicinal plants since primordial times as the fundamental source of medicines. The ethnomedicinal data related to folk medicines led to further exploration of medicinal plants as potential source of medicines resulting in the isolation of various natural products that have emerged as important pharmaceutics (Sharma and Mujundar, 2003). The most important characteristic of natural products contributing to their significance in drug discovery is their structural diversity (Veeresham, 2012). Secondary metabolites are biological entities that are not involved in the usual growth and development processes of the plants but play a major role in their defense system. These secondary metabolites including terpenes, phenolics and alkaloids are classified according to their biosynthetic origin. During the course of evolution diverse classes of these compounds have been associated with particular plant species and comprise the biologically active compounds in numerous therapeutically active medicinal and aromatic plants and useful foods (Roze et al., 2011). Alkaloids are a large and structurally diverse group of compounds which have a heterocyclic nitrogenous ring system and a basic (alkaline) character e.g., atropine and nicotine. Alkaloid producing plants are Abbreviations: CNS, central nervous system; DMSO, dimethylsulfoxide; IC50, inhibitory concentration with 50% inhibition; LB, Luria–Bertani; ppm, parts per million. ⁎ Corresponding author. E-mail address: [email protected] (Z. Saddiqe).
https://doi.org/10.1016/j.sajb.2018.05.031 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
commonly found in families like Fabaceae, Liliaceae, Solanaceae and Amaryllidaceae. Alkaloids are well known for their special pharmacological properties and are found in all plant parts like stems, roots, leaves, seeds, fruits and flowers. The literature reveals that alkaloids have diuretic, antitumor, antifungal, cardiotonic, antispermatogenetic, antiandrogenic, immunomodulatory, antipyretic and several effects on the central nervous system (CNS) (Patel et al., 2013). Cancer is a major public health problem in both developed and developing countries. The instability of cell growth and death can cause tumors to be formed (Rashed, 2014). It is an irregular development of body cells. Such growths can be malignant (cancerous) or benign (noncancerous). After cardiovascular disease, it is the world's second killer (Kathiriya et al., 2010). Tumors are believed to be triggered by the mutual action between genomic liability and environmental poisons. Medicinal plants have been established as a common substitute for synthetic drugs for tumor inhibition and treatment in various countries around the world. Natural phytochemicals obtained from medicinal plants have contributed significantly towards the treatment of several human diseases including cancer (Mehta et al., 2010). Over 3000 plants worldwide have been reported to possess anticancer properties (Dai and Mumper, 2010). The potato disc bioassay is a commonly used method to check the antitumor potential of plants and plant derived compounds. The assay is established on the basis of infection caused by Agrobacterium tumefaciens on potato discs (Islam et al., 2009). The basis for this bioassay is that the mechanism of tumor induction by A. tumefaciens is similar for both animals and plants (Becker, 1975).
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Antimitotic agents can modify or inhibit the process of cell division and thus are helpful in life threatening diseases like cancer (Gaikwad et al., 2011). The antimitotic activity can be tested using meristematic cells of Allium cepa root that are commonly used in testing of drugs for antimitotic activity. The growing tips of plant roots generally undergo repeated cell divisions and the rate of mitosis is higher in such regions compared to that of the other tissues. These cells can be used for initial screening of drugs with antitumor activity due to the presence of uniform meristematic cells, huge chromosomes and merely 16 chromosomes (Andrade et al., 2008; Thenmozhi et al., 2011). Datura alba Rumphius ex Nees belonging to the family Solanaceae is known for its medicinal uses. All parts of the plant have medicinal value, but only the leaves and seeds are formally used. It is extensively used for the treatment of asthma, healing of burn wounds, muscle spasm, whooping cough, hemorrhoids and skin ulcers etc. (Uddin et al., 2012). In the whole plant of Datura, many different alkaloids are found, which increase slowly but surely with increase in age of the plant. Major alkaloid components of D. alba comprise of large number of tropane alkaloids containing hyoscyamine, a number of withanolides, littorine, hyoscine, valtropine, acetoxytropine, fastusine and several tigloyl-esters of tropine and pseudotropine. Daturanolone, triterpene, β-sitosterol and daturadiol are mainly present in the fruit wall while the seeds predominantly contain tropane alkaloids along with daturanolone and fastusic acid. The nor-tropane alkaloids, calystegines, have also been found in a number of Datura species and are known to possess glycosidase inhibitory activity (Ghani, 2003). The ether extract of D. stramonium exhibited antitumor effects through inhibition of mitosis process (Ahmad et al., 2009). In a study by Nazeema Banu et al. (2014) the methanol extracts of leaves and stem of D. metel were evaluated for anticancer activity against MCF-7 cell lines. The study confirmed that the leaf extract had significant anticancer potential against MCF-7 cell lines than the stem extract. Several withanolides were isolated from the methanol extract of flowers of D. metel that were screened for inhibitory activity against various cancer cell lines and were found to be active with IC50 values ranging from 0.05 to 3.5 μM (Pan et al., 2007). On the basis of all these reports the total alkaloids from leaves of D. alba were extracted and were tested for antitumor and antimitotic activity. 2. Materials and methods 2.1. Collection and preparation of plant material Leaves of mature D. alba were collected from Lahore College for Women University. The plant was identified using Flora of Pakistan and other available resources and a voucher specimen was deposited in Prem Madan Herbarium (Voucher no. LCWU-18-233). The leaves were carefully separated from the plant, washed and surface sterilized. The leaf material was dried and crushed into powdered form to be used for further alkaloid extraction. 2.2. Extraction of total alkaloids Approximately 70 g of powdered leaf material was soaked in 200 mL of methanol for 5 days with constant stirring. The methanol extract was filtered and the process was repeated again in order to ensure that no extractables remained in the residues. The methanol was recovered by rotary evaporator at 40 °C. The residues from the methanol extracts were air dried and stored for further use. The filtrate was acidified with 0.1 M H2SO4 and extracted with chloroform. The organic phase-I contained neutral and acidic materials which was stored while the aqueous phase-I was tested for alkaloids with Dragendorff's reagent. This aqueous phase-I was basified with 20% Na2CO3 to pH 9/10 and again extracted with chloroform. The aqueous phase-II obtained contained the water soluble material whereas organic phase-II was
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washed with H2O and dried with Na2SO4. The chloroform was evaporated in a rotary at 40 °C. The latter solvent was completely dried up and the residue left was weighed to calculate the amount of crude alkaloids. The residue was tested with Dragendorff's reagent to confirm the presence of alkaloids. 2.3. Antitumor activity Antitumor activity of alkaloid extract of D. alba was determined by using the potato disc method as stated by Ferrigni et al. (1982). 2.3.1. Preparation of bacterial culture Culture of A. tumefaciens pathogenic strain (LBA4404) was grown on LB (Luria–Bertani) agar medium. LB medium was prepared by dissolving 1.25 g of LB in 50 mL of distilled water and pH was set at 7.0. It was autoclaved in 100 mL flask. Four to five isolated colonies from culture plate of A. tumefaciens were transferred to LB broth and incubated for 48 h at 30 °C. In a test tube, 10 ml of phosphate buffer with pH 7.2 was taken and about six to seven loops of bacterial suspensions were added in it. 2.3.2. Sample preparation Plant material was prepared by dissolving 10 mg of plant alkaloidal extract in 1 mL of DMSO (10 mg/mL or 10,000 ppm) to make stock solution. From this stock solution additional dilutions (1000 ppm, 100 ppm, and 10 ppm) were prepared. 2.3.3. Preparation of inoculum To prepare final concentration of 1000, 100 and 10 ppm, 1.5 mL of inoculum was prepared from initial stocks by adding 0.15 mL of each of the stock solution in three autoclaved test tubes. Finally 0.75 mL of double distilled (autoclaved) water and 0.60 mL culture of bacteria were added in each test tube. 0.15 mL of DMSO replacing alkaloid solution served as negative control while 0.15 mL of DMSO and 1.35 mL of double distilled (autoclaved) water served as blank. In order to avoid contamination, each solution was prepared in a Laminar flow hood and all precautionary measures were considered. 2.3.4. Preparation of agar plates To prepare the plane agar medium, 15 g/L of plane agar was dissolved in distilled water and sterilized in an autoclave. Three plates were used for each concentration (1000 ppm, 100 ppm, 10 ppm) and three plates for three controls. In each petri plate, about 20 mL of autoclaved agar solution was transferred and allowed to solidify. 2.3.5. Preparation of potato discs Red skinned potatoes were surface sterilized by dipping them in 0.1% HgCl 2 solution in a beaker for 10 min. The potato was then rinsed thrice with autoclaved, distilled water and dried. A sterilized borer was used to make cylinders of potato. The potato cylinders were splashed in distilled water in a petri plate. Thick discs (5 mm) of potato cylinders were made in petri plates. Potato discs were sterilized with autoclaved water and transferred on solidified agar plates (10 discs for each plate). On the surface of all discs, 50 μL of inoculum was poured. Within 10–20 min, the inoculum was diffused. The plates were made air-tight by wrapping them with parafilm. These petri-plates were put into an incubator at 28 °C for 21 days. 2.3.6. Staining of potato discs Lugol's solution was prepared in distilled water by adding 10% KI and 5% Iodine in it. The potato discs were coated with lugol's solution and allowed to diffuse for 15 min. The discs were examined under light microscope. The unstained portions of discs were the tumors. No. of
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tumors for each disc was calculated. The following formula was used to determine the percentage inhibition for respective concentrations. No:of tumors with extract 100 Percentage inhibition ¼ 100− No:of tumors with control More than 20% tumor inhibition was considered significant (Ferrigni et al., 1982). 2.4. Antimitotic assay Antimitotic activity of alkaloidal extract of D. alba was carried out by using the method described by Fiskesjo (1993). 2.4.1. Growing A. cepa meristems Healthy onion bulbs, after removal of dried outer scales, were placed in a series of jars containing normal tap water until 2–3 cm of roots were grown from each bulb. Tap water was changed periodically at an interval of 24 h. All the bulbs that did not show any sign of rooting were considered non-viable and were not used further in the study. 2.4.2. Conditions for plant extract incubation Working dilutions of crude plant alkaloids (10, 100 and 1000 ppm) were made in ethanol. The onion bulbs with roots measuring 2–3 cm were placed over alkaloid solution and incubated at room temperature for 24 h. The mitotic index was noted at 24 h and compared with bulbs placed over tap water that served as negative control. 2.4.3. Microscopic examination and determination of mitotic index After 72 h of treatment, the bulbs were taken out form the alkaloid solution. The tips of roots were cut with a sharp blade and transferred to the fixing solution containing acetic acid: ethanol (1:3) and then slightly heated in 1 N HCl. The treated root tips were then squashed and finally stained with methylene blue. For each root tip, hundred cells were counted in 5–8 fields. The slides were observed under high power (40 ×) of light microscope and the number of dividing and non-dividing cells was counted. In all cases cells with different stages of mitosis i.e., interphase, prophase (P), metaphase (M), anaphase (A) and telophase (T) were calculated. The following formula was used to calculate Mitotic index: Mitotic Index ¼
ðP þ M þ A þ TÞ Total cells
2.4.4. Statistical analysis The data was statistically analyzed using SPSS software and expressed as mean ± SEM. Least Significant Difference (LSD) test was used to speculate further if there was a significant difference. P values b0.05 were considered as significant.
Table 1 Extraction yield of crude methanol extract and alkaloid content of D. alba. Dry weight (g)
Crude methanol (g)
Alkaloid content (g)
70.03 % Yield
15.60 22.3%
0.272 1.74%
material and depends on the geographical area, part of the plant used and the growth stage (Vitale et al., 1995). The antitumor activity of alkaloidal fraction from D. alba leaves was tested through potato disc antitumor bioassay using A. tumefaciens. Alkaloidal leaf extract of D. alba inhibited the growth of crown gall tumors on potato discs in a dose dependent manner. Higher concentration of alkaloids gave significant inhibition of tumors on potato discs. At the highest concentration of 1000 ppm, strong antitumor activity was observed with 84.78% tumor inhibition, whereas the lowest alkaloid concentration of 10 ppm also gave significant result with 64.1% tumor inhibition (Figs. 1 and 2). IC50 value was calculated to be 10.7 ppm (Table 2). A. tumefaciens is a gram-negative bacterium that causes crown-gall infection in different plant species (Chilton et al., 1977). It is a neoplastic disease generated by Ti plasmid present in this bacterium (Tzfira et al., 2004). The Ti-plasmid comprises hereditary information termed as T-DNA which triggers self-directed tumors in plant species by means of erosions (Morris, 1986). T-DNA causes cell proliferation in plants without inducing apoptosis leading to tumor formation similar to cancer development in animals and humans (Zupan et al., 2000; Christie et al., 2005). Helicobacter pylori (Raderer et al., 1998) and Bartonella henselae (Kempf et al., 2002), the two bacteria that cause tumor formation in humans, have a similar pathogenicity strategy as adopted by A. tumefaciens (Zhu et al., 2000). Alkaloids are natural drugs that play chief role as antitumor agents by inhibiting the topoisomerase enzyme, which is directly involved in replication of DNA, causing apoptosis (Wall et al., 1966; Srivastava et al., 2005). A number of plant products such as vincristine, vinblastine and derivatives of podophyllotoxin including camptothecin and etoposide have previously been used as strong antitumor drugs (Jachak and Saklalni, 2007). The findings in the present study revealed that alkaloids from D. alba can be important bioactive components for separation of plant based anticancer compounds, which can be a good addition in plant-based anticancer drugs. Islam et al. (2008) reported the antitumor potential of twig methanol extract of Datura metel using potato disc bioassay through three different Agrobacterium strains. Highest inhibition of tumor (39.16%) was detected at concentration of 1000 ppm against the AtSl0105 strain. At 10 ppm, no tumor inhibition was observed. In our study the alkaloidal extract of D. alba leaves produced strong antitumor effect with 84.78% to 64.1% inhibition of tumor formation at the same concentrations. A. cepa test is a rapid, sensitive and reproducible bioassay for testing the antimitotic property of phytochemicals and the Allium test is suitable for both eukaryotes and prokaryotes (Levan, 1938). In the present study the alkaloid extract of plant leaves exhibited strong antimitotic 12
3. Results and discussion Average no. of tumors
Plants are continually providing us innovative chemical entities for the development of drugs against different diseases such as HIV/AIDS, cancer, Alzheimer's disease, pain and malaria. The quantity and quality of phytochemicals in any plant indicates the intensity of pharmacological and biological effect of that particular plant. The very first step in phytochemical study is to extract those active plant components from the plant part by applying an appropriate extraction method and choosing a proper solvent for that purpose. To obtain the crude extract, methanol was used as extracting solvent in the current study. Leaves of D. alba gave 22.3% of crude methanol extract and 1.74% of alkaloids were extracted from this crude extract (Table 1). The total alkaloid content in Datura varies from 0.02 to 0.52% relative to dry weight of plant
10
10 8 6
6
4.5
100ppm 10ppm
4 2
1000ppm
Control 1.5
0 concentraon mg/ml Fig. 1. Average number of tumors at different concentrations of the alkaloid extract.
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Fig. 2. Photographs showing tumor inhibition by alkaloid extract of D. alba on potato discs A) Bacterium (LBA4404) b and c) 10 ppm concentration d) 100 ppm e) 1000 ppm.
activity against A. cepa root tip cells. The results were obtained on the basis of mitotic index and are presented in Table 3 and in Fig. 3. A. cepa roots gave a reduced mitotic index percentage, reflecting the effect of alkaloid content on meristematic root cells. The results were compared with the water control in which 95% mitotic index was observed (Fig. 3). Roots grown in the control solution (water) displayed actively dividing cells at different mitotic stages i.e., prophase, metaphase, anaphase and telophase (Fig. 5). The dose-dependent decrease in mitotic index revealed the antimitotic activity of alkaloids. The leaf alkaloid extract of D. alba inhibited the meristematic cell division in A. cepa root tips in a dose dependent manner. As compared to control group (P ˂ 0.05), higher alkaloid concentrations significantly lowered mitotic index. At the highest concentration of 1000 ppm, strong antimitotic activity was noticed for the alkaloid extract with a mitotic index of 30 ± 0.577 while least active was the 10 ppm concentration of alkaloid with high mitotic index of 91 ± 1.529. The cells showing division marked the presence of prophase stage, while other stages of division i.e., metaphase, anaphase and telophase were somewhat rare.
Alkaloids possessing antimitotic activity such as vinblastine and paclitaxel work by activating the spindle assembly checkpoint (SAC) and ultimately prevent polymerization of microtubules (Masawang et al., 2014). Microtubules are structures developed during interphase and are essential for normal cell division and segregation of chromosomes. The rate of formation of microtubules is faster during mitosis as compared to interphase, and hence microtubules are considered as best drug targets as cancer cells generally have high rate of proliferation (Dumontet and Jordan, 2010). The antimitotic drugs block transition of cells from metaphase to anaphase which results in mitotic arrest in cells. These drugs also affect formation of spindle and positioning of chromosome on the spindle causing the cells to remain either in G1 phase or in a persistent condition of arrest thus leading to apoptosis (Mitchison, 2012). Noscapine, a phthalide-isoquinoline alkaloid extracted from latex of opium, is a nontoxic antitussive agent that attaches to tubulin, obstructs cell division in mitosis and causes apoptosis and therefore has been established as an antimitotic agent (Dhiman et al., 2013).
Table 2 Percentage inhibition of tumor formation at 3 different concentrations.
Datura alba
Percentage tumor inhibition at concentrations 1000 ppm
100 ppm
10 ppm
84.78
76.1
64.1
120 IC50 ppm
100 10.7
Table 3 Antimitotic activity of alkaloid extract of D. alba. Concentration (ppm)
Total no. of cells
No. of non-dividing cells
P
M
A
T
Total dividing cells
Mitotic index (%)
Control (water) 10 ppm 100 ppm 1000 ppm
100 100 100 100
4 8 19 70
85 80 78 30
5 3 – –
3 5 – –
2 3 – –
95 91 78 30
95 ± 1.527a 91 ± 1.529b 78 ± 0.578c 30 ± 0.577d
P = Prophase; M = Metaphase; A = Anaphase; T = Telophase. Values with different alphabets are significantly different from each other at P = 0.05.
Mitoc index (%)
Alkaloid extract
80 60 40 20 0 Control (water)
10ppm
100ppm
1000ppm
concentraon (ppm) Fig. 3. Mitotic index (%) for control and different concentrations of Alkaloid extract.
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Fig. 4. A. cepa root tips in control (A) and in alkaloid extract (B).
Fig. 5. Normal mitotic stages 1) interphase, 2) prophase, 3) metaphase, 4) anaphase and 5) telophase.
Fig. 6. Nuclear and chromosomal abnormalities A) Binucleolar and nucleolar burst, B) Stickiness, C) Condensed anaphase, D) Abnormal metaphase, E) Chromosomal clumping in cells, F) Spindle disturbance, G) C-metaphase, H) Condensed anaphase, I) Anaphase bridge, J) Disturbed spindle, K) Chromosomal bridge, L) C-metaphase M) Telophase laggard.
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3.1. Morphological and mitotic abnormalities There were no mitotic abnormalities detected in control root tips. They showed normal mitosis stages including prophase, metaphase, anaphase and telophase. However, meristematic cells of onion root tips exposed to the plant alkaloid extract displayed chromosomal and morphological abnormalities. Root tips dipped in alkaloid extract tend to have a brown color with broken tips, while healthy tips are white in color without any damage (Fig. 4). The alkaloid extract of D. alba caused abnormalities in the process of mitosis as well as in the structure of chromosomes involving spindle disturbance at prophase, chromosomal clumping, stickiness in small cells, abnormal metaphase, nucleolar and binucleolar burst (Fig. 6). 4. Conclusion The results of the present study indicated that the alkaloid content of D. alba leaves showed significant antitumor and antimitotic potential and thus can be further studied for its anticancer activity using cancer cell lines. References Ahmad, I., Abdalla, M., Mustafa, N., Qnais, E., Abdalla, F., 2009. Datura aqueous leaf extract enhance cytotoxicity via metabolic oxidative stress on different human cancer cells. Jordon Journal of Biological Sciences 2, 9–14. Andrade, L.F., Campos, J.M.S., David, L.C., 2008. Cytogenetic alterations induced by SPL (spent pot-liners) in meristematic cells of plant bioassays. Ecotoxicology and Environmental Safety 71, 706–710. Becker, F.F., 1975. Cancer: a comprehensive treatise. Etiology: Viral Carcinogenesis vol. 2. Plenum Press, pp. 350–355. Chilton, M.D., Drummond, M.H., Merio, D.J., Sciaky, D., Montoya, A.L., Gordon, M.P., Nester, E.W., 1977. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11, 263–271. Christie, P.J., Atmakuri, K., Krishnamoorthy, V., Jakubowski, S., Cascales, E., 2005. Biogenesis, architecture, and function of bacterial type IV secretion systems. Annual Reviews in Microbiology 59, 451–485. Dai, J., Mumper, R.J., 2010. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules 15, 7313–7352. Dhiman, N., Sood, A., Sharma, A., 2013. Noscapine: an anti-mitotic agent. World Journal of Pharmacy and Pharmaceutical Sciences 3, 324–338. Dumontet, C., Jordan, M.A., 2010. Microtubule binding agents: a dynamic field of cancer therapeutics. Natural Reviews Drug Discovery 9, 790–803. Ferrigni, N.R., Putnam, J.E., Anderson, B., Jacobsen, L.B., Nichols, D.E., Moore, D.S., McLaughlin, J.L., Powell, R.G., Smith Jr., C.R., 1982. Modification and evaluation of the potato disc assay and antitumor screening of Euphorbiaceae seeds. Journal of Natural Products 45, 679–686. Fiskesjo, G., 1993. Allium test I: a 2–3 day plant test for toxicity assessment by measuring the mean root growth of onions (Allium cepa L.). Environmental Toxicology and Water Quality 8, 461–470. Gaikwad, S.B., Mohan, K.G., Anerthe, S.J., 2011. Antimitotic activity and brine shrimp lethality test of Tectona grandis Linn, bark. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2, 1014–1022. Ghani, A., 2003. Medicinal Plants of Bangladesh with Chemical Constituents and Uses. second edition. Asiatic Society of Bangladesh, 5 old Secretariat Road, Nimtali, Dhaka, Bangladesh. Islam, M.S., Rehman, M.M., Alam, M.J., Nurunnahar, Pervez, M.S., Anisuzzaman, M., Alam, M.F., 2008. Screening of antitumor activity of Datura metel L., using potato disc bioassay. Plant Environmental Development 2, 87–92.
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Alkaloid rich fraction of Datura alba Rumph. ex Nees leaves possesses antitumor and antimitotic activity Z. Saddiqe ⁎, U. Wahab, A. Maimoona, R. Raheel, M. Iram Department of Botany, Lahore College for Women University, Lahore, Pakistan
a r t i c l e
i n f o
Article history: Received 15 December 2017 Received in revised form 26 April 2018 Accepted 30 May 2018 Available online xxxx Edited by JJ Nair Keywords: Agrobacterium tumefaciens Allium cepa root tip Alkaloids Antimitotic Antitumor Datura alba
a b s t r a c t The antimitotic and antitumor activities of total alkaloids extracted from leaves of Datura alba Nees. were investigated using in vitro methods. The plant leaves were collected from local ground of Lahore College for Women University. The antitumor bioassay was performed using potato discs applied with plant alkaloid extract and activity was checked against Agrobacterium tumefaciens. The results demonstrated that the alkaloid rich fraction significantly repressed the tumor growth on potato discs at a concentration of 1000 ppm, giving 84.78% tumor inhibition after 21 days of incubation. The antimitotic bioassay was implemented using Allium cepa root tip bioassay. The findings suggested that the alkaloidal fraction also showed significant antimitotic effect on root tip cells of A. cepa. At 1000 ppm concentration, the alkaloidal extract revealed best antimitotic activity giving reduced (30 ± 0.577%) mitotic index after 72 h of treatment. The outcome of the present experiment, therefore, indicates that Datura alkaloids could be regarded as a potential source for the development of anti-tumor and antimitotic agents. The study could be extended further for drug development against cancers in humans. © 2018 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Human beings have been utilizing medicinal plants since primordial times as the fundamental source of medicines. The ethnomedicinal data related to folk medicines led to further exploration of medicinal plants as potential source of medicines resulting in the isolation of various natural products that have emerged as important pharmaceutics (Sharma and Mujundar, 2003). The most important characteristic of natural products contributing to their significance in drug discovery is their structural diversity (Veeresham, 2012). Secondary metabolites are biological entities that are not involved in the usual growth and development processes of the plants but play a major role in their defense system. These secondary metabolites including terpenes, phenolics and alkaloids are classified according to their biosynthetic origin. During the course of evolution diverse classes of these compounds have been associated with particular plant species and comprise the biologically active compounds in numerous therapeutically active medicinal and aromatic plants and useful foods (Roze et al., 2011). Alkaloids are a large and structurally diverse group of compounds which have a heterocyclic nitrogenous ring system and a basic (alkaline) character e.g., atropine and nicotine. Alkaloid producing plants are Abbreviations: CNS, central nervous system; DMSO, dimethylsulfoxide; IC50, inhibitory concentration with 50% inhibition; LB, Luria–Bertani; ppm, parts per million. ⁎ Corresponding author. E-mail address: [email protected] (Z. Saddiqe).
https://doi.org/10.1016/j.sajb.2018.05.031 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
commonly found in families like Fabaceae, Liliaceae, Solanaceae and Amaryllidaceae. Alkaloids are well known for their special pharmacological properties and are found in all plant parts like stems, roots, leaves, seeds, fruits and flowers. The literature reveals that alkaloids have diuretic, antitumor, antifungal, cardiotonic, antispermatogenetic, antiandrogenic, immunomodulatory, antipyretic and several effects on the central nervous system (CNS) (Patel et al., 2013). Cancer is a major public health problem in both developed and developing countries. The instability of cell growth and death can cause tumors to be formed (Rashed, 2014). It is an irregular development of body cells. Such growths can be malignant (cancerous) or benign (noncancerous). After cardiovascular disease, it is the world's second killer (Kathiriya et al., 2010). Tumors are believed to be triggered by the mutual action between genomic liability and environmental poisons. Medicinal plants have been established as a common substitute for synthetic drugs for tumor inhibition and treatment in various countries around the world. Natural phytochemicals obtained from medicinal plants have contributed significantly towards the treatment of several human diseases including cancer (Mehta et al., 2010). Over 3000 plants worldwide have been reported to possess anticancer properties (Dai and Mumper, 2010). The potato disc bioassay is a commonly used method to check the antitumor potential of plants and plant derived compounds. The assay is established on the basis of infection caused by Agrobacterium tumefaciens on potato discs (Islam et al., 2009). The basis for this bioassay is that the mechanism of tumor induction by A. tumefaciens is similar for both animals and plants (Becker, 1975).
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Antimitotic agents can modify or inhibit the process of cell division and thus are helpful in life threatening diseases like cancer (Gaikwad et al., 2011). The antimitotic activity can be tested using meristematic cells of Allium cepa root that are commonly used in testing of drugs for antimitotic activity. The growing tips of plant roots generally undergo repeated cell divisions and the rate of mitosis is higher in such regions compared to that of the other tissues. These cells can be used for initial screening of drugs with antitumor activity due to the presence of uniform meristematic cells, huge chromosomes and merely 16 chromosomes (Andrade et al., 2008; Thenmozhi et al., 2011). Datura alba Rumphius ex Nees belonging to the family Solanaceae is known for its medicinal uses. All parts of the plant have medicinal value, but only the leaves and seeds are formally used. It is extensively used for the treatment of asthma, healing of burn wounds, muscle spasm, whooping cough, hemorrhoids and skin ulcers etc. (Uddin et al., 2012). In the whole plant of Datura, many different alkaloids are found, which increase slowly but surely with increase in age of the plant. Major alkaloid components of D. alba comprise of large number of tropane alkaloids containing hyoscyamine, a number of withanolides, littorine, hyoscine, valtropine, acetoxytropine, fastusine and several tigloyl-esters of tropine and pseudotropine. Daturanolone, triterpene, β-sitosterol and daturadiol are mainly present in the fruit wall while the seeds predominantly contain tropane alkaloids along with daturanolone and fastusic acid. The nor-tropane alkaloids, calystegines, have also been found in a number of Datura species and are known to possess glycosidase inhibitory activity (Ghani, 2003). The ether extract of D. stramonium exhibited antitumor effects through inhibition of mitosis process (Ahmad et al., 2009). In a study by Nazeema Banu et al. (2014) the methanol extracts of leaves and stem of D. metel were evaluated for anticancer activity against MCF-7 cell lines. The study confirmed that the leaf extract had significant anticancer potential against MCF-7 cell lines than the stem extract. Several withanolides were isolated from the methanol extract of flowers of D. metel that were screened for inhibitory activity against various cancer cell lines and were found to be active with IC50 values ranging from 0.05 to 3.5 μM (Pan et al., 2007). On the basis of all these reports the total alkaloids from leaves of D. alba were extracted and were tested for antitumor and antimitotic activity. 2. Materials and methods 2.1. Collection and preparation of plant material Leaves of mature D. alba were collected from Lahore College for Women University. The plant was identified using Flora of Pakistan and other available resources and a voucher specimen was deposited in Prem Madan Herbarium (Voucher no. LCWU-18-233). The leaves were carefully separated from the plant, washed and surface sterilized. The leaf material was dried and crushed into powdered form to be used for further alkaloid extraction. 2.2. Extraction of total alkaloids Approximately 70 g of powdered leaf material was soaked in 200 mL of methanol for 5 days with constant stirring. The methanol extract was filtered and the process was repeated again in order to ensure that no extractables remained in the residues. The methanol was recovered by rotary evaporator at 40 °C. The residues from the methanol extracts were air dried and stored for further use. The filtrate was acidified with 0.1 M H2SO4 and extracted with chloroform. The organic phase-I contained neutral and acidic materials which was stored while the aqueous phase-I was tested for alkaloids with Dragendorff's reagent. This aqueous phase-I was basified with 20% Na2CO3 to pH 9/10 and again extracted with chloroform. The aqueous phase-II obtained contained the water soluble material whereas organic phase-II was
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washed with H2O and dried with Na2SO4. The chloroform was evaporated in a rotary at 40 °C. The latter solvent was completely dried up and the residue left was weighed to calculate the amount of crude alkaloids. The residue was tested with Dragendorff's reagent to confirm the presence of alkaloids. 2.3. Antitumor activity Antitumor activity of alkaloid extract of D. alba was determined by using the potato disc method as stated by Ferrigni et al. (1982). 2.3.1. Preparation of bacterial culture Culture of A. tumefaciens pathogenic strain (LBA4404) was grown on LB (Luria–Bertani) agar medium. LB medium was prepared by dissolving 1.25 g of LB in 50 mL of distilled water and pH was set at 7.0. It was autoclaved in 100 mL flask. Four to five isolated colonies from culture plate of A. tumefaciens were transferred to LB broth and incubated for 48 h at 30 °C. In a test tube, 10 ml of phosphate buffer with pH 7.2 was taken and about six to seven loops of bacterial suspensions were added in it. 2.3.2. Sample preparation Plant material was prepared by dissolving 10 mg of plant alkaloidal extract in 1 mL of DMSO (10 mg/mL or 10,000 ppm) to make stock solution. From this stock solution additional dilutions (1000 ppm, 100 ppm, and 10 ppm) were prepared. 2.3.3. Preparation of inoculum To prepare final concentration of 1000, 100 and 10 ppm, 1.5 mL of inoculum was prepared from initial stocks by adding 0.15 mL of each of the stock solution in three autoclaved test tubes. Finally 0.75 mL of double distilled (autoclaved) water and 0.60 mL culture of bacteria were added in each test tube. 0.15 mL of DMSO replacing alkaloid solution served as negative control while 0.15 mL of DMSO and 1.35 mL of double distilled (autoclaved) water served as blank. In order to avoid contamination, each solution was prepared in a Laminar flow hood and all precautionary measures were considered. 2.3.4. Preparation of agar plates To prepare the plane agar medium, 15 g/L of plane agar was dissolved in distilled water and sterilized in an autoclave. Three plates were used for each concentration (1000 ppm, 100 ppm, 10 ppm) and three plates for three controls. In each petri plate, about 20 mL of autoclaved agar solution was transferred and allowed to solidify. 2.3.5. Preparation of potato discs Red skinned potatoes were surface sterilized by dipping them in 0.1% HgCl 2 solution in a beaker for 10 min. The potato was then rinsed thrice with autoclaved, distilled water and dried. A sterilized borer was used to make cylinders of potato. The potato cylinders were splashed in distilled water in a petri plate. Thick discs (5 mm) of potato cylinders were made in petri plates. Potato discs were sterilized with autoclaved water and transferred on solidified agar plates (10 discs for each plate). On the surface of all discs, 50 μL of inoculum was poured. Within 10–20 min, the inoculum was diffused. The plates were made air-tight by wrapping them with parafilm. These petri-plates were put into an incubator at 28 °C for 21 days. 2.3.6. Staining of potato discs Lugol's solution was prepared in distilled water by adding 10% KI and 5% Iodine in it. The potato discs were coated with lugol's solution and allowed to diffuse for 15 min. The discs were examined under light microscope. The unstained portions of discs were the tumors. No. of
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tumors for each disc was calculated. The following formula was used to determine the percentage inhibition for respective concentrations. No:of tumors with extract 100 Percentage inhibition ¼ 100− No:of tumors with control More than 20% tumor inhibition was considered significant (Ferrigni et al., 1982). 2.4. Antimitotic assay Antimitotic activity of alkaloidal extract of D. alba was carried out by using the method described by Fiskesjo (1993). 2.4.1. Growing A. cepa meristems Healthy onion bulbs, after removal of dried outer scales, were placed in a series of jars containing normal tap water until 2–3 cm of roots were grown from each bulb. Tap water was changed periodically at an interval of 24 h. All the bulbs that did not show any sign of rooting were considered non-viable and were not used further in the study. 2.4.2. Conditions for plant extract incubation Working dilutions of crude plant alkaloids (10, 100 and 1000 ppm) were made in ethanol. The onion bulbs with roots measuring 2–3 cm were placed over alkaloid solution and incubated at room temperature for 24 h. The mitotic index was noted at 24 h and compared with bulbs placed over tap water that served as negative control. 2.4.3. Microscopic examination and determination of mitotic index After 72 h of treatment, the bulbs were taken out form the alkaloid solution. The tips of roots were cut with a sharp blade and transferred to the fixing solution containing acetic acid: ethanol (1:3) and then slightly heated in 1 N HCl. The treated root tips were then squashed and finally stained with methylene blue. For each root tip, hundred cells were counted in 5–8 fields. The slides were observed under high power (40 ×) of light microscope and the number of dividing and non-dividing cells was counted. In all cases cells with different stages of mitosis i.e., interphase, prophase (P), metaphase (M), anaphase (A) and telophase (T) were calculated. The following formula was used to calculate Mitotic index: Mitotic Index ¼
ðP þ M þ A þ TÞ Total cells
2.4.4. Statistical analysis The data was statistically analyzed using SPSS software and expressed as mean ± SEM. Least Significant Difference (LSD) test was used to speculate further if there was a significant difference. P values b0.05 were considered as significant.
Table 1 Extraction yield of crude methanol extract and alkaloid content of D. alba. Dry weight (g)
Crude methanol (g)
Alkaloid content (g)
70.03 % Yield
15.60 22.3%
0.272 1.74%
material and depends on the geographical area, part of the plant used and the growth stage (Vitale et al., 1995). The antitumor activity of alkaloidal fraction from D. alba leaves was tested through potato disc antitumor bioassay using A. tumefaciens. Alkaloidal leaf extract of D. alba inhibited the growth of crown gall tumors on potato discs in a dose dependent manner. Higher concentration of alkaloids gave significant inhibition of tumors on potato discs. At the highest concentration of 1000 ppm, strong antitumor activity was observed with 84.78% tumor inhibition, whereas the lowest alkaloid concentration of 10 ppm also gave significant result with 64.1% tumor inhibition (Figs. 1 and 2). IC50 value was calculated to be 10.7 ppm (Table 2). A. tumefaciens is a gram-negative bacterium that causes crown-gall infection in different plant species (Chilton et al., 1977). It is a neoplastic disease generated by Ti plasmid present in this bacterium (Tzfira et al., 2004). The Ti-plasmid comprises hereditary information termed as T-DNA which triggers self-directed tumors in plant species by means of erosions (Morris, 1986). T-DNA causes cell proliferation in plants without inducing apoptosis leading to tumor formation similar to cancer development in animals and humans (Zupan et al., 2000; Christie et al., 2005). Helicobacter pylori (Raderer et al., 1998) and Bartonella henselae (Kempf et al., 2002), the two bacteria that cause tumor formation in humans, have a similar pathogenicity strategy as adopted by A. tumefaciens (Zhu et al., 2000). Alkaloids are natural drugs that play chief role as antitumor agents by inhibiting the topoisomerase enzyme, which is directly involved in replication of DNA, causing apoptosis (Wall et al., 1966; Srivastava et al., 2005). A number of plant products such as vincristine, vinblastine and derivatives of podophyllotoxin including camptothecin and etoposide have previously been used as strong antitumor drugs (Jachak and Saklalni, 2007). The findings in the present study revealed that alkaloids from D. alba can be important bioactive components for separation of plant based anticancer compounds, which can be a good addition in plant-based anticancer drugs. Islam et al. (2008) reported the antitumor potential of twig methanol extract of Datura metel using potato disc bioassay through three different Agrobacterium strains. Highest inhibition of tumor (39.16%) was detected at concentration of 1000 ppm against the AtSl0105 strain. At 10 ppm, no tumor inhibition was observed. In our study the alkaloidal extract of D. alba leaves produced strong antitumor effect with 84.78% to 64.1% inhibition of tumor formation at the same concentrations. A. cepa test is a rapid, sensitive and reproducible bioassay for testing the antimitotic property of phytochemicals and the Allium test is suitable for both eukaryotes and prokaryotes (Levan, 1938). In the present study the alkaloid extract of plant leaves exhibited strong antimitotic 12
3. Results and discussion Average no. of tumors
Plants are continually providing us innovative chemical entities for the development of drugs against different diseases such as HIV/AIDS, cancer, Alzheimer's disease, pain and malaria. The quantity and quality of phytochemicals in any plant indicates the intensity of pharmacological and biological effect of that particular plant. The very first step in phytochemical study is to extract those active plant components from the plant part by applying an appropriate extraction method and choosing a proper solvent for that purpose. To obtain the crude extract, methanol was used as extracting solvent in the current study. Leaves of D. alba gave 22.3% of crude methanol extract and 1.74% of alkaloids were extracted from this crude extract (Table 1). The total alkaloid content in Datura varies from 0.02 to 0.52% relative to dry weight of plant
10
10 8 6
6
4.5
100ppm 10ppm
4 2
1000ppm
Control 1.5
0 concentraon mg/ml Fig. 1. Average number of tumors at different concentrations of the alkaloid extract.
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Fig. 2. Photographs showing tumor inhibition by alkaloid extract of D. alba on potato discs A) Bacterium (LBA4404) b and c) 10 ppm concentration d) 100 ppm e) 1000 ppm.
activity against A. cepa root tip cells. The results were obtained on the basis of mitotic index and are presented in Table 3 and in Fig. 3. A. cepa roots gave a reduced mitotic index percentage, reflecting the effect of alkaloid content on meristematic root cells. The results were compared with the water control in which 95% mitotic index was observed (Fig. 3). Roots grown in the control solution (water) displayed actively dividing cells at different mitotic stages i.e., prophase, metaphase, anaphase and telophase (Fig. 5). The dose-dependent decrease in mitotic index revealed the antimitotic activity of alkaloids. The leaf alkaloid extract of D. alba inhibited the meristematic cell division in A. cepa root tips in a dose dependent manner. As compared to control group (P ˂ 0.05), higher alkaloid concentrations significantly lowered mitotic index. At the highest concentration of 1000 ppm, strong antimitotic activity was noticed for the alkaloid extract with a mitotic index of 30 ± 0.577 while least active was the 10 ppm concentration of alkaloid with high mitotic index of 91 ± 1.529. The cells showing division marked the presence of prophase stage, while other stages of division i.e., metaphase, anaphase and telophase were somewhat rare.
Alkaloids possessing antimitotic activity such as vinblastine and paclitaxel work by activating the spindle assembly checkpoint (SAC) and ultimately prevent polymerization of microtubules (Masawang et al., 2014). Microtubules are structures developed during interphase and are essential for normal cell division and segregation of chromosomes. The rate of formation of microtubules is faster during mitosis as compared to interphase, and hence microtubules are considered as best drug targets as cancer cells generally have high rate of proliferation (Dumontet and Jordan, 2010). The antimitotic drugs block transition of cells from metaphase to anaphase which results in mitotic arrest in cells. These drugs also affect formation of spindle and positioning of chromosome on the spindle causing the cells to remain either in G1 phase or in a persistent condition of arrest thus leading to apoptosis (Mitchison, 2012). Noscapine, a phthalide-isoquinoline alkaloid extracted from latex of opium, is a nontoxic antitussive agent that attaches to tubulin, obstructs cell division in mitosis and causes apoptosis and therefore has been established as an antimitotic agent (Dhiman et al., 2013).
Table 2 Percentage inhibition of tumor formation at 3 different concentrations.
Datura alba
Percentage tumor inhibition at concentrations 1000 ppm
100 ppm
10 ppm
84.78
76.1
64.1
120 IC50 ppm
100 10.7
Table 3 Antimitotic activity of alkaloid extract of D. alba. Concentration (ppm)
Total no. of cells
No. of non-dividing cells
P
M
A
T
Total dividing cells
Mitotic index (%)
Control (water) 10 ppm 100 ppm 1000 ppm
100 100 100 100
4 8 19 70
85 80 78 30
5 3 – –
3 5 – –
2 3 – –
95 91 78 30
95 ± 1.527a 91 ± 1.529b 78 ± 0.578c 30 ± 0.577d
P = Prophase; M = Metaphase; A = Anaphase; T = Telophase. Values with different alphabets are significantly different from each other at P = 0.05.
Mitoc index (%)
Alkaloid extract
80 60 40 20 0 Control (water)
10ppm
100ppm
1000ppm
concentraon (ppm) Fig. 3. Mitotic index (%) for control and different concentrations of Alkaloid extract.
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Fig. 4. A. cepa root tips in control (A) and in alkaloid extract (B).
Fig. 5. Normal mitotic stages 1) interphase, 2) prophase, 3) metaphase, 4) anaphase and 5) telophase.
Fig. 6. Nuclear and chromosomal abnormalities A) Binucleolar and nucleolar burst, B) Stickiness, C) Condensed anaphase, D) Abnormal metaphase, E) Chromosomal clumping in cells, F) Spindle disturbance, G) C-metaphase, H) Condensed anaphase, I) Anaphase bridge, J) Disturbed spindle, K) Chromosomal bridge, L) C-metaphase M) Telophase laggard.
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3.1. Morphological and mitotic abnormalities There were no mitotic abnormalities detected in control root tips. They showed normal mitosis stages including prophase, metaphase, anaphase and telophase. However, meristematic cells of onion root tips exposed to the plant alkaloid extract displayed chromosomal and morphological abnormalities. Root tips dipped in alkaloid extract tend to have a brown color with broken tips, while healthy tips are white in color without any damage (Fig. 4). The alkaloid extract of D. alba caused abnormalities in the process of mitosis as well as in the structure of chromosomes involving spindle disturbance at prophase, chromosomal clumping, stickiness in small cells, abnormal metaphase, nucleolar and binucleolar burst (Fig. 6). 4. Conclusion The results of the present study indicated that the alkaloid content of D. alba leaves showed significant antitumor and antimitotic potential and thus can be further studied for its anticancer activity using cancer cell lines. References Ahmad, I., Abdalla, M., Mustafa, N., Qnais, E., Abdalla, F., 2009. Datura aqueous leaf extract enhance cytotoxicity via metabolic oxidative stress on different human cancer cells. Jordon Journal of Biological Sciences 2, 9–14. Andrade, L.F., Campos, J.M.S., David, L.C., 2008. Cytogenetic alterations induced by SPL (spent pot-liners) in meristematic cells of plant bioassays. Ecotoxicology and Environmental Safety 71, 706–710. Becker, F.F., 1975. Cancer: a comprehensive treatise. Etiology: Viral Carcinogenesis vol. 2. Plenum Press, pp. 350–355. Chilton, M.D., Drummond, M.H., Merio, D.J., Sciaky, D., Montoya, A.L., Gordon, M.P., Nester, E.W., 1977. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11, 263–271. Christie, P.J., Atmakuri, K., Krishnamoorthy, V., Jakubowski, S., Cascales, E., 2005. Biogenesis, architecture, and function of bacterial type IV secretion systems. Annual Reviews in Microbiology 59, 451–485. Dai, J., Mumper, R.J., 2010. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules 15, 7313–7352. Dhiman, N., Sood, A., Sharma, A., 2013. Noscapine: an anti-mitotic agent. World Journal of Pharmacy and Pharmaceutical Sciences 3, 324–338. Dumontet, C., Jordan, M.A., 2010. Microtubule binding agents: a dynamic field of cancer therapeutics. Natural Reviews Drug Discovery 9, 790–803. Ferrigni, N.R., Putnam, J.E., Anderson, B., Jacobsen, L.B., Nichols, D.E., Moore, D.S., McLaughlin, J.L., Powell, R.G., Smith Jr., C.R., 1982. Modification and evaluation of the potato disc assay and antitumor screening of Euphorbiaceae seeds. Journal of Natural Products 45, 679–686. Fiskesjo, G., 1993. Allium test I: a 2–3 day plant test for toxicity assessment by measuring the mean root growth of onions (Allium cepa L.). Environmental Toxicology and Water Quality 8, 461–470. Gaikwad, S.B., Mohan, K.G., Anerthe, S.J., 2011. Antimitotic activity and brine shrimp lethality test of Tectona grandis Linn, bark. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2, 1014–1022. Ghani, A., 2003. Medicinal Plants of Bangladesh with Chemical Constituents and Uses. second edition. Asiatic Society of Bangladesh, 5 old Secretariat Road, Nimtali, Dhaka, Bangladesh. Islam, M.S., Rehman, M.M., Alam, M.J., Nurunnahar, Pervez, M.S., Anisuzzaman, M., Alam, M.F., 2008. Screening of antitumor activity of Datura metel L., using potato disc bioassay. Plant Environmental Development 2, 87–92.
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