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. 2011 Aug;1(4):295–297. doi: 10.1016/S2221-1691(11)60046-4

Studies on isozymic variation among the South Indian species of Sphaerostephanos

Irudayaraj Varaprasadham 1, Johnson Marimuthu 1,*
PMCID: PMC3614243  PMID: 23569778

Abstract

Objective

To explore the identity and phylogenetic relationships among the three medicinally important species of Sphaerostephanos from South India using isozymic profile.

Methods

The young fronds were homogenized with 3.5 mL of ice-cold homogenizing buffer in a pre-chilled pestle and mortar. The supernatant was subjected to electrophoresis as described by Anbalagan poly acrylamide gel electrophoresis. Staining solutions for isoperoxidase was prepared as per Smila method for the detection of isoenzymes.

Results

A total of six different bands in five different positions with different molecular weight/Rf values and four active zones have been observed in the isoperoxidase enzyme system of Sphaerostephanos. Only one band with MW/Rf 0.399 is common to two different species i.e. Sphaerostephanos arbuscula (S. arbuscula) and Sphaerostephanos unitus (S. unitus). Among the remaining four bands, two bands (Rf. 0.23, 0.47) are present in Sphaerostephanos subtruncatus (S. subtruncatus) and one distinct band has been observed individually in S. arbuscula (Rf. 0.507) and S. unitus (Rf. 0.56).

Conclusions

The present preliminary molecular study through isozymic analysis shows the identity of all the three species and the present results confirm distinctness of these three species based on macro-micromorphology, phytochemistry and cytology.

Keywords: Sphaerostephanos, Isoperoxidase, Poly acrylamide gel electrophoresis, Inter-specific variation , Isozymic variation, Isozymic profile, Phylogenetic relationship

1. Introduction

The primitive vascular plants pteridophytes form the dominant vegetation on the earth next to flowering plants. The megabiodiversity country India is rich in pteridophytes with the presence of about 1 200 species distributed in various taxonomical families. Several species of pteridophytes are medicinally important and they are used by local people to treat various kinds of health problems. The South Indian thelypteroid genus Sphaerostephanos is represented by three distinct species i.e. Sphaerostephanos arbuscula (Willd.) Holttum (S. arbuscula), Sphaerostephanos subtruncatus (Bory) Holttum (S. subtruncatus) and Sphaerostephanos unitus (L.) Holttum (S. unitus). In general species of Sphaerostephanos are used to treat various health problems. The crushed young leaves are used to rub on scabies. Alternatively, the leaves are boiled and the juice is used to bathe the patient suffering from scabies. For skin conditions associated with measles, the new leaves and shoots are squeezed and the juice is rubbed on the affected area. The plant is also used in treating fever[1]. Yusuf et al[2] made the chemotaxonomic survey of flavonoids from Sphaerostephanos (Thelypteridaceae) of Peninsular Malaysia. From S. arbuscula the medicinally important chemical compound astilbin has been reported[3]. Astilbin prevents concanavalin A-induced liver injury by reducing TNF-α production and T lymphocyte adhesion[4]. The inhibitory effect of astilbin on the arteriosclerosis of murine thoracic aorta transplant has been studied by Zhao et al[5]. Astilbin also suppresses acute heart allograft rejection by inhibiting maturation and function of dendritic cells in mice[6]. Song et al[7] have also explored the effects of astilbin on the maturation and immunologic function of mouse bone marrow-derived dendritic cells. Antibacterial activity has been proved both in S. subtruncatus and S. unitus[8],[9]. Identity of any medicinally important species is essential in the modern world. They are distinct in macromorphology (frond, rhizome) and micromorphology (morphology and distribution of epidermal glands)[10]. Chemical composition of epidermal glands is also different among these three species[11]. Both S. unitus and S. arbuscula are diploid sexual species with 36 bivalents in Spore Mother Cells[12]. In contrast, S. subtruncatus is a tetraploid species (n=72). Thus cytologically S. arbuscula and S. unitus are similar while S. subtruncatus is distinct. However, in chemical composition of epidermal glands, S. arbuscula and S. subtruncatus are more or less similar in having less chemical diversity-eight compounds in each species while S. unitus is distinct in having higher degree of chemical diversity (17 different compounds). Based on morphology, cytology and phytochemistry, it is very difficult to identify the medicinal plants in the form of crude drug. Molecular marker is very useful in pharmacognosy. Since the differences in all the morphological and phytochemical characters are ultimately due to the difference in genetical characters, the study on genetical difference may be useful for identity and exploration of phylogenetic relationships among the three medicinally important species of Sphaerostephanos from South India. In contrast to DNA marker, isoenzyme analyses are widely used for their relative efficiency and cost- effectiveness, particularly in studies of intra and inter-specific variability[13]. Isozymes are as useful as genetic and biochemical markers and also as good estimators of genetic variability in plant populations. With this background, the differences in the isozymic profile have been analysed in the present study.

2. Materials and methods

Plants S. arbuscula (Willd.) Holttum, S. subtruncatus (Bory) Holttum and S. unitus (L.) Holttum were collected from the natural habitats and established in the green house attached to the Centre for Biodiversity and Biotechnology, St. Xavier's College (Autonomous), Palayamkottai, Tamil Nadu, India. From the mother plants, croziers were collected and homogenized with 3.5 mL of ice-cold homogenizing buffer in a pre-chilled pestle and mortar. The supernatant was subjected to electrophoresis as described by Anbalagan[14] on poly acrylamide gel electrophoresis (PAGE). Staining solutions for isoperoxidase was prepared as per Smila[15] for the detection of iso-enzymes. After the electrophoresis, the gels were incubated in the staining solution for a few minutes under dark condition till the clear bands appeared. The gels were fixed with 7% acetic acid solution for 30 min, washed with distilled water and photographed using the gel documentation system manufactured by Biotech, Yercaud, India. Based on the isoenzyme banding profile the zymogram was constructed.

3. Results

A total of six different bands in five different positions with different molecular weight/Rf values and four active zones have been observed in the isoperoxidase enzyme system of Sphaerostephanos (Figure 1). Only one band was observed in zone three PRX31 (0.230) and it was restricted to S. subtruncatus. Zone four expressed with two bands in one position PRX41 (0.399) and jointly expressed in S. unitus and S. arbuscula. Zone five also showed only one band PRX51 (0.470); and it is present only in S. subtruncatus. The other two species failed to express any band in the zone five. Two bands were observed in zone six. PRX61 (0.507) was expressed only in S. arbuscula and PRX62 (0.560) was present only in S. unitus (Table 1).

Figure 1. Zymogram of the isoperoxidase profile of Sphaerostephanos spp.

Figure 1.

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S. a-S. arbuscula; S. s-S. subtruncatus; S. u-S. unitus.

Table 1. Isoperoxidase profile of Sphaerostephanos spp.

MW/Rf Value S. arbuscula S. subtruncatus S. unitus
0.230 +
0.399 + +
0.470 +
0.507 +
0.560 +
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4. Discussion

All the three south Indian species are similar in having two different bands in each species. But they differ in having bands of different molecular weight and Rf values. Only one band with MW/Rf 0.399 is common to two different species, i.e. S. arbuscula and S. unitus. Among the remaining four bands, two bands (Rf. 0.230, 0.470) are present in S. subtruncatus and one distinct band has been observed individually in S. arbuscula (Rf. 0.507) and S. unitus (Rf. 0.560). Thus the present preliminary molecular study through isozymic analysis shows the identity of all three species and the present results confirm distinctness of these three species based on macro-micromorphology, phytochemistry and cytology. The two diploid species share the band with Rf value of 0.399 which is absent in the tetraploid species S. subtruncatus. S. arbuscula is both morphologically and cytologically primitive in having erect rhizome and with diploidal chromosome number (n=36)[16]–[19]. S. subtruncatus is morphologically primitive and cytologically advanced in having erect rhizome and tetraploidal chromosome number (n=72) and S. unitus is morphologically advanced and cytologically prmitive in having long creeping rhizome and diploidal chromosome number (n=36). The same kind of trend is also observed in the present molecular study in having different bands with different molecular weight and different Rf values. Thus the distinct banding patterns of the presently studied medicinal plants will be useful for the pharmacognostical identification of the crude drugs of the same species in the near future. The present study results were directly coincided with previous observations[20]–[28].Further studies on molecular taxonomy on these species will give more knowledge about the molecular identity and phylogenetic relationship among these three South Indian species of Sphaerostephanos.

Footnotes

Conflict of interest statement: We declare that we have no conflict of interest.

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