INTRODUCTION:

Plantago ovata (Family: Plantaginaceae) is an annual herb that grows in sandy regions of the mediterranean basin, India, Iran, North and northwest Africa, Pakistan and Southern Europe. Psyllium is the common name used for several members of the plant genus Plantago whose seeds are used commercially for the production of mucilage. The genus Plantago comprises over 200 species. P. ovata and P. psyllium are produced commercially in several European and Asian countries. Isabgol, the common name in India for P. ovata, comes from the Persian words "isap" and "ghol" that mean ‘horse ear’, which is descriptive of the shape of the seed.

The seeds and husk of Plantago ovata are used in traditional and modern systems of medicine. Seeds are cooling, useful in inflammatory and bilious derangement of digestive organs, good in dysentry and irritation of the intestinal tract, decoction in cough and chronic diarrhoea. The husk from the seeds has the property of absorbing and retaining water and hence, it is used for treatment of diarrhea ¹.

Psyllium is produced mainly for its mucilage content, which is highest in P. ovata. Mucilage obtained from psyllium comes from the seed coat. So, Psyllium is a soluble fiber used primarily as a gentle bulk laxative. In vitro cytotoxic, antiviral and immunomodulatory effect of Plantago major and Plantago asiatica had been examined and concluded that the hot water extracts of P. major and P. asiaticia possessed a broad-spectrum of antileukaemia, anticarcinoma and antiviral activities².

MATERIAL AND METHODS:

To obtain Stock solution, 1gm husk of Plantago ovata is dissolved in 1000 ml of distilled water. The required ascending concentrations of 100ppm, 250ppm, 500ppm, 750ppm and 1000ppm were prepared by serial dilution method from Stock solution. The Vicia faba seeds were presoaked for the 12 hrs. in water and then grown in sterile moist sand at 30⁰C to 35⁰C for obtaining the secondary roots by the technique³. First of all, roots were cut without any treatment to find control value of mitotic index for Vicia faba root meristem. The roots were then treated with different concentrations of Plantago ovata husk aqueous solution for two hours, four hours and six hours and after that roots were cut carefully and then fixed in Carnoy’s solution (3 ethanol: 1 Acetic acid) for 24 hrs. and transferred to 70% alcohol for preservation. The root tips had been hydrolyzed in 1N HCl for 5 minutes and squashed in 2% acetocarmine for cytological studies. The slides were temporarily sealed and examined. The mitotic index had been calculated using the method⁴. Chromosomal aberrations and their percentage in each treatment were recorded. Chromosomal aberrations were photographed microscopically. To examine recovery, treated roots were left in distilled water for 24 hrs and mitotic index was recorded.

RESULTS:

All the concentrations of P. ovata husk aqueous suspension inhibited the mitotic activity of Vicia root tip cells. The percentage value of mitotic index decreased gradually with an increase in concentration and duration of the treatment (Table-1 and Photoplate-1). The observed mitotic index for Vicia root tip cells in controlled condition was 22.82%. When the root tips were treated with 100ppm aqueous suspension of husk, the mitotic index reduced to 18.71% (2 hours), 18.60% (4 hours) and 16.73% (6 hours). The mitotic index further decreased to 16.95% (2 hours), 16.75% (4 hours) and 16.66% (6 hours) by the treatment of 250 ppm concentration of medicinal plant husk. The treatment of 500ppm had maintained the declinity in percentage value of mitotic index and recorded mitotic index was 16.19% (2 hours), 15.50% (4 hours) and 15.19% (6 hours). Mito-inhibition continued in 750ppm treatment and mitotic index dropped to 15.10% (2 hours), 14.38% (4 hours) and 14.37% (6 hours). The downfall in mitotic index of V. faba root apical cells treated by 1000ppm aqueous suspension of P. ovata husk was noticed as 14.56% (2 hours), 11.57% (4 hours) and 11.04% (6 hours) (Table 1).

The recovered mitotic index of V. faba root apical cells treated with all the used concentrations of P. ovata husk was increased after a recovery period of 24 hours duration. Maximum recovery was recorded in 500ppm treatment for 2 hours and minimum recovery was in 750ppm treatment for 2 hours duration (Table 1).

Binucleate cells were observed at interphase when roots were treated by lower concentrations viz. 100ppm and 250 ppm only and the recorded percentage of binculeate cells was 0.21% and 0.22% respectively (Table 1).

At prophase, none of the aberration was observed. Chromosomal aberrations such as breakage, clumping, fragmentation of chromosomes, ring formation, scattered metaphase and stickiness of chromosomes were recorded during metaphase. The value of chromosomal breakage was 13.88%, 5.00%, 11.11% and 5.26% in the treatments of 100ppm, 250ppm, 750ppm and 1000ppm, respectively and no chromosomal breakage was reported in 500ppm treatment. Clumping of chromosomes was induced during all treatments above 100ppm aqueous concentration of P. ovata husk and its recorded mean percent values were 5.00% (250ppm), 4.00% (500ppm), 5.55% (750ppm) and 5.26% (1000ppm), chromosome fragments were observed during treatment of 500ppm for 4 hours only and its value was 4.00%. The concentrations of 100ppm and 750ppm only were potentially capable of inducing 2.77% and 5.55% ring formation, respectively. Scattered metaphase was most prominent anomaly at metaphase and it is interesting to note that percent value of chromosomal scattering increased with increased concentration of husk aqueous suspension. Thus, the mean percent values i.e. 5.55%, 7.50%, 8.00%, 16.66% and 21.05% were directly proportional to the used concentrations i.e. 100ppm, 250ppm, 500ppm, 750ppm and 1000ppm respectively in the treatments. The treatments by 500ppm and 750ppm concentrations only induced 4.00% and 16.66% stickiness of chromosomes, respectively (Photoplate-1).

Mitotic anomalies observed at anaphase were bridge formation, chromosomal extrusion and polarity abolition. Bridge formation was observed during treatment by 250ppm for 2 hours only and recorded value was 5.26%. All the used concentrations except 500ppm induced extrusion of chromosome and its recorded values were 21.42% (100ppm), 5.26% (250ppm), 16.66% (750ppm) and 27.27% (1000ppm). The polarity abolition was caused by higher concentrations in a dose dependent manner. The polarity abolition was 5.55% in 500 ppm, increased to 11.11% (750ppm) and reached to 18.18% in 1000ppm treatment (Table -1 and Photoplate-1). No mitotic anomaly was noticed at telophase.

DISCUSSION:

Herbal medicines have a long history of use in prevention and treatment of diseases⁵ but in contrast with conventional drug research and development, the toxicity of traditional herbal medicines is not often evaluated. In the post-genome era, several in silico, in vitro and in vivo approaches and methods could be applied to predict genotoxicity and teratogenicity of herbal medicinal products^{6, 7}.

In the recent past, much attention has been paid to record the cytotoxic, mutagenic and genototoxic effects of vegetables, spices, drugs, alkaloids, dyes and other chemical substances such as herbicides, pesticides and insecticides. However, a fragmentary work is available on medicinal plants. So, the present study has been done to evaluate the cytogenetic effect of certain medicinal plants on chromosomal behavior during somatic divisions in plant test system. Investigator has tried her best to test the cytotoxicity of undertaken medicinal plants and to explain its possible reasons with the facilities available. The investigator does not claim herself to be perfect and complete in the discussion of the subject but has an opinion that the present study will definitely open many new approaches for research.

Chromosome breakage has been observed in V. faba root tip chromosomes treated with some medicinal plant extracts⁸. Chromosome breaks were also reported in V. faba treated by trifluralin⁹. The occurrence of chromosome fragments was reported in A. sativum root tip cells treated with extract of castor seed¹⁰and also observed in V. faba root meristems while investigating mutagenic activities of uccmaluscide¹¹. Similar fragments were also noticed in A. cepa treated by dinocap¹². Rings have also been reported while studying the effect of water extract from Boerrhavia diffusa roots on Helianthus annus¹³. Ring chromosomes are frequently formed in eukaryotes as a result of change in chromosomal configuration. Normal chromosomes do not form rings because they have telomeres on each end and these telomers prevent the union of chromosomes arms to form rings. A chromosome can form ring by fusion of the broken ends, only if it has two terminal deletions producing a centric segment with two raw ends and two acentric fragments. The ring chromosome inherits the centromere and the terminally deleted acentric fragment can unite into an acentric fragment that eventually gets lost from the nucleus. A ring chromosome is devoid of genetic information carried by the terminally deleted acentric fragments. Generally, ring chromosomes are mitotically unstable. During mitosis they can produce two daughter rings of equal size that are regularly distributed to the daughter cells. Such ring chromosomes can be somatically stable¹⁴. Ring chromosomes usually result from two terminal breaks in both chromosome arms, followed by fusion of the broken ends, or from the union of one broken chromosome end with the opposite telomere region, leading to the loss of genetic material and the clinical phenotype of patients with ring chromosomes may be related with different factors, including gene haplo-insufficiency, gene duplications and ring instability¹⁵.

Aqueous husk suspension of Plantago were found to be potentially capable of inducing scattering of chromosomes at metaphase in Vicia faba root meristem cells. (Table 1). It is interesting to note, scattering was caused by Plantago in Vicia in a dose dependent manner. Scattering of chromosomes was observed in cultured cells of Hemerocallis hybrid¹⁶. Sticky chromosomes were found while analyzing dose response of medicinal plant Viola odorata on meiotic and mitotic chromosomes of Vicia faba¹⁷. Sticky chromosomes, fragments and anaphasic bridges were found while assessing mitodepressive and clastogenic effects of aqueous extracts of the lichens Myelochroa lindmanii and Canoparmelia texana (Lecanorales, Parmeliaceae) on meristematic cells in plant bioassays¹⁸. Stickiness usually leads to the formation of anaphase and telophase bridges and this end up inhibiting metaphase and cytokinesis respectively and thus hampering cell division. Stickiness might be due to the ability of the extracts to cause DNA depolymerization and partial dissolution of nucleoproteins, breakage and exchanges of the basic folded units of chromatids and the stripling of the protein covering of DNA in chromosomes as also observed¹⁹. Anaphasic bridges were also observed in somatic cells of A. cepa treated with fungicide afugan²⁰ and with dinocap¹². In V. faba, bridges were found following treatment of chlorobenzene ²¹ and of some medicinal plant extracts⁸. Bridges were resulted from difficulty in anaphase separation due to adhesion or clumping of chromosomes²² or it is formed by depolymerisation and cross-linking of DNA of chromosomes²³. It has been observed that bridges are formed by attachment of sister chromosomes at the secondary constriction resulted in the fusion of bridges leading to a high occurrence of single and multiple bridges²⁴. Ultimately these bridges were broken up and the broken ends withdrew into the resting nuclei, in which these were often present in pear-shaped projection to mark their position. The bridges may lead to loss of genetic material of the cell²⁵. Chromosomal instability occurs early in the development of cancer and may represent an important step in promoting the multiple genetic changes required for the initiation and/or progression of the disease²⁶. Telomere erosion is one of the factors that contribute to chromosome instability through end-to-end chromosome fusions entering BFB (breakage–fusion–bridge) cycles. Uncapped chromosomes with short dysfunctional telomeres represent an initiating substrate for both pre- and post-replicative joining, which leads to unstable chromosome rearrangements prone to bridge at mitotic anaphase. Resolution of chromatin bridge intermediates is likely to contribute greatly to the generation of segmental chromosome amplification events, unbalanced chromosome rearrangements and whole chromosome aneuploidy. Accordingly, telomere-driven instability generates highly unstable genomes that could promote cell immortalization and the acquisition of a tumour phenotype. Mitodepression by aqueous suspension of Plantago husk also supports its cytotoxic effects. Cytotoxic and genotoxic effects of aqueous extracts of five medicinal plants have been observed on Allium cepa²⁷. Carbofuran induced cytogenetic effects in root meristem cells of Allium cepa and Allium sativum²⁸. Mutagenic and antimutagenic potentials of fruit juices of five medicinal plants were examined in Allium cepa²⁹. Cytological studies have been done in Allium root meristem by treating extract of medicinal plant Orthosiphon thymiflorus and chromosomal aberrations as micronuclei, bridges, fragments, laggards, multipolar anaphase, stickiness, ring chromosomes and extrusion³⁰. Thus the mito-inhibition and chromosomal aberrations caused by aqueous suspension of Plantago ovata in Vicia faba root meristem revealed cytotoxic potentiality of aqueous suspension of Plantago ovata husk in root meristem of Vicia faba and therefore sought to explore its cytotoxic potentiality in animal system with a view of its safe use in folkloric medicine. Furthermore, plant cytotoxic bioassays have a good correlation with mammalian systems^31,32,33,34.

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Received on 02.04.2017 Modified on 27.04.2017

Research J. Pharm. and Tech. 2017; 10(7): 2053-2057.

DOI: 10.5958/0974-360X.2017.00358.4


A. Fragmentation of chromosomes at metaphase	B. Scattered metaphase	C. Stickiness of chromosomes at metaphase

D. Chromosomal extrusion at anaphase	E. Polarity abolition at anaphase	F.Ring formation at metaphase

PHOTOPLATE -1 (A-F): Chromosomal aberrations in V. faba root meristem cells as induced by aqueous suspesion of Plantago ovata husk.