Bryophytes are the second largest group of plants after the flowering plants. About 20,000 members were documented from all over the world. Out of which 3000 bryophytes were reported to possess medicinal properties. They are primitive non-vascular plants and include liverworts, hornworts and mosses as three distinct groups. These plants were traditionally used in Chinese, European and Indian medicine to treat various illness including skin diseases and bronchitis. Numerous biologically active compounds were reported from these plants such as phenolic compounds, alkaloids, flavonoids and terpenoids. Plagiochila beddomei is a liverwort reported to possess the phenolic acids like coumaric acid, Ferulic acid, gallic acid, caffeic acid, cinnamic acid, sinapic acid and hydroxyl benzoic acid.1 There is no authentic historical record, describing the application of liverworts and mosses in Ayurveda, the Indian system of medicine. Interestingly, mosses such as Barbula, Fissidenc, Minium, Thuidium and species of liverworts like Asterella, Dumortiera, Marchantia, Pellia, Plagiochasma and Stephenrencella-Anthoceros were revealed the occurrence of minerals and metals in their thallus like copper, silver, zinc, iron, lead etc.2
Chinese ethnic usage revealed 40 kinds of bryophytes employed in cardiovascular diseases, tonsillitis, bronchitis, cystitis, and skin infections. Similarly, Fissidens and Polytrichum were utilized as diuretic and hair growth stimulating drugs long back of 400 years ago. Marchantía polymorpha was employed in curing liver diseases like jaundice and hepatitis. In China, Rhodobryum giganteum and R. roseum were used in the treatment of heart ailments.3
Bryum, Mnium, Philonotis spp., and Polytrichum juniperinum were used by the North American, Indians to heal burns, bruises, and wounds.The Seminole people in North America were reported to use Barbula unguiculata as medicinal. Similarly, Bryum capillare and Octoblepharum albidum were used as febrifuge and antidote against poison. Willow moss, Fontinalis antipyretica was reputed as a febrifuge. Dried Sphagnum was used extensively as a surgical dressing material during the First World War. In France, Marchantia polymorpha was used to promote diuresis. In the Himalayas, Riccia sp. was used for the treatment of Tinea (ringworm) infestations. The use of bryophytes as antibacterial or disinfectant agents deserves special mention. Sphagnum teres was used in ophthalmologic diseases. In China and Bolivia, Fissidens osmundoides was used as an antibacterial agent to treat inflammatory conditions of the pharynx and larynx. Haplocladium microphyllum was used as a demulcent medicine in inflammatory conditions like bronchitis, cystitis, tonsillitis and tympanitis. Philonotis fontana was used by Go suite native people as a soothing preparation for healing burns.2
Chetna Sharma et al.4 reported that the phenolic compounds present in the methanolic extract of Reboulia hemispherica exhibited microbicidal potential against Staphylococcus aureus indicating their therapeutic potentiality. Hari Datta et al.5 reported Sanionia uncinata a polar moss as an important source of natural antioxidant agents and Boris pejin et al.6 reported the moss Bryum moravicum possess potential antioxidant activity. Similarly, the ethanolic extracts of Atrichum undulatum, Polythrichum formosum (Polytrichaceae) possess stronger antioxidant activity.7 Fifilyana et al.8 reported Sphagnum cuspidatum subsp. subrecurvum, Sphagnum cuspidatulum, Sphagnum junghuhniannum, Pogonatum cirratum subsp. fuscatum and Pogonatum cirratum subsp. macrophyllum contained considerable amount of phenolics and flavonoids which contribute to their antioxidant properties.
Fourier Transformer Infra-red (FTIR) Spectrometry is also a valuable tool for the identification of biologically active compounds based on their functional groups and chemical compounds present in the plant extracts. But, still the experimental evidence regarding their usage among bryophytes is not fully proved. So, in this scenario, the aim of the present study was to identify the functional groups present in the various solvent extracts of Thuidium tamariscellum by FTIR analysis and also to evaluate the antioxidant potentiality of the moss.
MATERIALS AND METHODS
Thuidium tamariscellum (Thuidiaceae) identity was confirmed by floras and authenticated by comparing with the herbarium of University of Calicut.
Estimation of terpenoids
Total terpenoids was determined by the method of Ferguson.9
DPPH Radical Scavenging Assay
2, 2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) antioxidant method as described by Blois10 was used to analyze the free radical scavenging ability of the moss and the absorbance was measured at 517 nm. Ascorbic acid was used as the standard.
ABTS•+ radical scavenging assay
The 2,2’-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay 1 is based on the oxidation of the ABTS by potassium persulfate to form a radical cation 2,2’-azino-bis-3- ethylbenzothiazoline-6-sulphonic acid (ABTS+). The absorbance was taken at 734 nm and the activity was expressed as percentage inhibition of 2, 2’-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radicals.
Hydrogen peroxide radical
A solution of 40 mM H2O2 was prepared in phosphate buffer (pH-7.4). 1.4 ml of different concentrations of the extracts was added to 0.6 ml of the H2O2 solution. The assay mixture could stand for 10 min at 25°C and the absorbance was measured against a blank solution at 230 nm.12
Metal chelating activity
The chelation of ferrous ions by terpenoids extract was estimated. The absorbance of the solution was measured at 562 nm. The percentage inhibition of ferrozine–Fe2+ complex formation was calculated as [(A0- As)/ A0] X 100, where A0 was the absorbance of the control, and as was the absorbance of the extract/ standard.13
The FRAP (Ferric reducing antioxidant power assay) was assayed as per the protocol of Benzie and Strain.14 The absorbance of the reaction mixture was read at 593 nm spectrophotometrically after incubation at 37ºC for 10 min.
FTIR spectrometer was used to collect spectra of different solvent extracts of the moss.15 The sample was mixed with KBr. The IR spectrum gives information about the functional groups. 4000 to 300 cm-1 is the range of measurements. The region above 1200 cm-1 shows spectral bands due to vibrations of individual functional groups whereas the region below1200 cm-1 shows bands due to vibrations of whole molecule, and is known as the fingerprint region.
RESULTS AND DISCUSSION
Primary and secondary metabolite screening of T. tamariscellum revealed a tremendous range of biologically active compounds such as carbohydrates, lipid, protein, steroids, phenols, terpenoids, organic acids, sugar alcohols, fatty acids, aliphatic compounds, acetogenins, phenylquinones, and aromatic and phenolic substances which substantiates its significant bioactivities. The plant derived natural products occupy an important place in life style diseases such as cancer chemotherapy with minimal side effects.
Total terpenoid content
Subsequently, the total terpenoid content was analyzed. Significant level of terpenoid was noticed in the moss T. tamariscellum i.e., 25.95 mg/g.
DPPH scavenging effect
This is one of the most common method used to analyze the free radical scavenging ability of the plant extracts. It is a stable free radical that accepts an electron in the presence of AOX, with a subsequent discolouration of the violet colour. The discoloration was stechiometrically related to the number of electrons accepted. A concentration dependent antioxidant activity was noticed with the terpenoid extract of the moss. The IC50 value of the extract was 16 µg/ml. The following moss species such as Sphagnum cuspidatum subsp. subrecurvum, Sphagnum cuspidatulum, Sphagnum junghuhniannum, Pogonatum cirratum subsp. fuscatum and Pogonatum cirratum subsp. macrophyllum displyayed considerable DPPH scavenging property.8 The present data was at par with that of the moss species previously reported.
Hydrogen peroxide scavenging
The moss terpenoid extract showed a concentration dependent scavenging of the hydroxyl radical and the IC50 value was 34.5 µg/ml. Hydrogen peroxide is basically a week oxidising agent, can cross cell membranes and react with ions like Fe2+ to form hydroxyl radical which is a strong oxidising agent and is toxic to the cell. This shows the moss extract can effectively scavenge hydroxyl radical. The values were statistically significant at 5% level.
ABTS radical scavenging activity
The terpenoid extract effectively scavenged the ABTS radicals in a dose dependent manner. At 30µg/ml the inhibition was 65.45% and that of ascorbate was 79.5%. The IC50 value of ascorbate was 10µg/ml while that of moss terpenoid extract was 18.5 µg/ml.
Fifilyana et al.8 reported that the aqueous, methanolic and ethanolic extract of the moss Pogonatum. cirratum subsp. fuscatum possess strong ABTS radical scavenging activity.
Ferric reducing antioxidant power (FRAP) assay
FRAP assay estimate the reducing potential of moss extract by reacting with a ferric tripyridyltriazine (Fe3+-TPTZ) complex to form a coloured ferrous tripyridyltriazine (Fe2+-TPTZ). Table 1 shows the ferric reducing power increased with an increase in concentration of the terpenoid extract. The IC50 of the terpenoid extract was 40 µg/ml while that of the synthetic antioxidant ascorbate was at 28 µg/ml.
Homalothecium sericeum and Eurhynchium striatulum possess stronger ferric reducing power 36.91 and 33.59 μmol/100g.16
Metal chelating activity
Table 1 shows a dose dependent increase in ferrous (Fe2+) metal ion chelation. This property is mainly due to the presence of endogenous chelating agents like terpenoids in the plant extract. 40 μg/ml concentration of the extract showed 55.28% chelation of ferrous ions. The IC50 value of the moss terpenoid extract was at 35µg/ml while that of ascorbate was at 22 µg/ml.
Wojtunik et al.17 proposed a model on the antioxidant potential of terpenoids constituents in 2,2-diphenyl-1-picrylhydrazyl method. Thoppil et al.18 reviewed the role of terpenoids as chemopreventive and therapeutic against liver cancer. Zwenger and Basu19 evaluated the roles and future potentials of plant terpenoids. Partap and pandey20 compared herbal antioxidants of a medicinal species. Nagarajan and Brindha21 compared diterpenes therapeutic uses with special emphasis on antidiabetic activity. Topçu et al.22 analyzed antioxidant potential of triterpenoids from Salvia macrochlamys. Kasote et al.23 proved remarkable nature of polyphenolics in terms of antioxidant potential to cure many life style diseases. Polyphenolic molecules contain one or many aromatic rings with hydroxyl groups. Generally, the antioxidant capacity of the phenolics is directly related with the number of free hydroxyls and conjugation of side chains with the aromatic rings.24 Flavonoids and phenolic acids of plant phenolics, are metabolically originated from the acetate via shikimate pathways, as well as the shikimate pathway from phenylalanine or tyrosine.25 Phytochemicals of this group are excellent antioxidants under in vitro and in vivo experimentals. In addition, they are proven to interplay with other natural antioxidants like ascorbate or tocopherol and to synergistically enhance their biological potentials.26 Flavonoids and phenylopropanoid derivatives are also oxidized by peroxidase, and act as H2O2 scavengers.27 In vitro experimental conditions showed that the antioxidant power of polyphenolics is always linked to their electron donation, reducing power and metal ion chelating ability.28 In this juncture, the present results were more significance in the sense that most of the proven works were from phenols or flavanoids. In the present study, the crude terpenoid content of the moss was high and displayed high antioxidant potentials.
The FTIR spectroscopic analysis of four different solvent extracts of T. tamariscellum showed the presence of various functional groups indicating the presence of bioactive compounds especially terpenes and terpenoids in the plant. Terpenoids were present with C=O stretch in 1714.72 cm-1 in petroleum ether extract, 1726.29 cm-1 in ethyl acetate, chloroform and methanol extract and with O-H stretch in 3409.29, 3456.44 and 3431.36 cm-1 in petroleum ether extract and 3404.36, 3454.51, 3431.3 cm-1 in methanol extract (Table 2). The presence of terpenes were also revealed due to C-H stretch in 2953.02, 2918.30, 2850.79 in petroleum ether extract, 2920.23 in ethyl acetate extract, 2922.16, 2854.65 in chloroform extract and 2931.80, 2854.65 in methanolic extract (Figure 1a,b,c,d).
Hydroxyl group (~3400cm-1) or an oxo group (saturated 1750-1700 cm-1) indicates the presence of terpenoids.29 The peak at 2926.01 cm-1 in the ethanolic extract of Tylophora pauciflora indicating the C-H stretching of alkane compounds in the rare medicinal plant.30 In the methanolic extract of Rhapis excelsa C-H stretch at 2856.7- 2927.1 cm-1 also reveals terpenoids in terms of C=O stretch at 1704.18, 1708.04 cm-1.31
The phytochemical study showed the presence of significant level of terpenoid in the moss which was validated by FTIR analysis showing peaks specific for terpenoids. Thus, the high antioxidant property shown by the plant is mainly due to the presence of considerable amount of terpenoids. Further studies are warranted to isolate the potential lead molecule and to evaluate its biological properties.
FTIR: Fourier Transformer Infra-red; AOX: Antioxidant; ABTS: 2,2’-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid assay; DPPH: 2,2-diphenyl-1-picryl-hydrazyl-hydrate.
CONFLICT OF INTEREST
The authors declare that there are no conflict of interest pertaining to this work.
The moss Thuidium tamariscellum is rich in secondary metabolites especially terpenoids. This data was validated by FTIR analysis showing the presence of various functional groups indicating the presence of terpenes and terpenoids. Further, the effective scavenging of DPPH, ABTS, FRAP, Hydrogen peroxide and metal chelating activity by the terpenoid extract from the moss clearly establishes the antioxidant power of the bryophyte.