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Evaluation of the immunomodulatory and DNA protective activities of the shoots of Cynodon dactylon
Journal of Ethnopharmacology 123 (2009) 181–184
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Evaluation of the immunomodulatory and DNA protective activities of the shoots of Cynodon dactylon K. Mangathayaru a,∗ , M. Umadevi b , C. Umamaheswara Reddy a a Sri Ramachandra College of Pharmacy, Sri Ramachandra University, Porur, Chennai 600 116, India b RVS College of Pharmaceutical Sciences, Sulur, Coimbatore 641 402, India
a r t i c l e
i n f o
Article history: Received 28 June 2007 Received in revised form 29 September 2008 Accepted 13 February 2009 Available online 4 March 2009 Keywords: Cynodon dactylon Immunomodulatory DNA protective Rasayana drug
a b s t r a c t Ethnopharmacological relevance: Fresh juice of Cyanodon dactylon known as ‘durva’ grass is employed in India as a rejuvenator and for wound healing. Aim of the study: To validate the traditional use of the herb through evaluation of DNA protective activity in vitro and immunomodulatory activity in vivo. Materials and methods: Fresh juice of the grass was prepared as indicated for use in traditional medicine and standardized for solid content. Its total phenol content was estimated by Folin–Ciocalteau method. Freshly prepared juice was investigated for its effect on doxorubicin-induced DNA damage in vitro. Its immunomodulatory activity was tested on balb/c mice by the humoral antibody response which was determined by haemagglutination antibody titer and spleen cell assay. Results: Fresh juice of Cyanodon dactylon of 1.46% (w/w) solid content had a phenolic content of 47 ± 0.33 mg/kg GAE. At doses equivalent to 50, 100 and 200 mg total solids/kg body weight the juice protected human DNA against doxorubicin-induced DNA damage as demonstrated in DNA spectral studies, where the ratio of absorbance of DNA at 260 and 280 nm in samples pretreated with the juice was 1.66, 1.53 and 1.63 respectively, while it was 1.37 for DNA treated with doxorubicin only. This indicates nucleic acid purity in the Cynodon dactylon treated samples. Oral administration of the juice at 250 and 500 mg/kg in balb/c mice increased humoral antibody response upon antigen challenge, as evidenced by a dose-dependent, statistically significant increase in antibody titer in the haemagglutination antibody assay and plaque forming cell assay. Conclusions: The present report demonstrated the DNA protective activity and immunomodulatory property of the fresh juice of Cynodon dactylon validating the traditional use of the herb as a ‘rasayana’ in ayurvedic system of medicine. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The xenobiotic modulation of immune response is of current scientific interest due to its potential in the treatment of immunocompromised conditions (Thatte and Dahanukar, 1986). Currently available immunosuppressive and immunostimulating agents have major limitations, such as increased risk of infection and inhibition of cells formed by the bone marrow (Diasio and Lo Buglio, 1996).
Several ayurvedic medicinal plants are powerful immunomodulators (Devasagayam and Saims, 2002). Recent research has linked their therapeutic actions to their strong antioxidant potential (Govindarajan et al., 2005). Despite their therapeutic importance, very few of them have been investigated in detail. The objective of this work is to evaluate a standardized fresh juice of Cynodon dactylon with respect to DNA protective as well as immunomodulatory effect in view of the use of the herb in neurodegenerative and immunological disorders. 1.1. Plant
Abbreviations: ACD, acid–citrate–dextrose; EDTA, ethylenediaminetetraacetic acid; TE, tris–ethylenediaminetetraacetic acid; TAE, tris–acetic acid–ethylenediaminetetraacetic acid; SRBC, sheep red blood cells; GAE, gallic acid equivalent; DNA, deoxyribonucleic acid; HPTLC, high performance thin layer chromatography; NaCl, sodium chloride; KCl, potassium chloride; MgCl2 , magnesium chloride. ∗ Corresponding author. Tel.: +91 24768403x8930. E-mail address: [email protected] (K. Mangathayaru). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.02.036
Cynodon dactylon Linn. Pers. (Poaceae) was collected from in and around Chennai and authenticated by Prof. P. Jayaraman, Director of Plant Anatomy Research Centre, Chennai, India. A voucher specimen (COPG-PG-A13) was deposited in the herbarium of the Department of Pharmacognosy, Sri Ramachandra College of Pharmacy, Sri Ramachandra University.
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K. Mangathayaru et al. / Journal of Ethnopharmacology 123 (2009) 181–184
1.2. Uses in traditional medicine A hardy perennial commonly called Bermuda grass, Cynodon dactylon is one of the most commonly occurring weeds in India. A ‘Rasayana’ drug, it is an inseparable part of religious rituals and is a valuable herbal medicine used as first aid in minor injuries (Oudhia, 1999). In Indian households it is a common practice to place strands of this grass on eatables during eclipse. Folk wisdom claims it to be water purifier (Artizzu et al., 1996). The juice of the plant is astringent and the fresh juice is used in the treatment of chronic diarrhea and dysentery (The Wealth of India, 2001). The plant occupies a renowned position in ayurveda, unani and homoeopathic systems of medicine (Chopra et al., 1956). Wound healing (Subramanian and Nagarajan, 1988), antimicrobial (Ahmed et al., 1994), antioxidant (Auddy et al., 2003) and estrogenic (Miller, 1967) activities of the plant are earlier reported. 1.3. Previously isolated classes of constituents Compounds including arundoin, furfural, furfuralcohol, ionone, 2-(4 -hydroxyphenyl)-propionic, 4-hydroxybenzoic, 2-(3 methoxy-4-hydroxyphenyl) propionic, and 3-methoxy-4-hydroxybenzoic acids, phytol, -sitosterol-d-glucoside, stigmasterol acetate and phytone have been isolated from Cynodon dactylon (Miller, 1967; Ottmoto et al., 1970; Rizk et al., 1986; Artizzu et al., 1996). 2. Materials and methods 2.1. Preparation of fresh juice The fresh shoots of Cynodon dactylon (1 kg) were washed, airdried and ground in a food processor and filtered. The juice so prepared as indicated for use in traditional medicine (Nadkarni, 1976) was standardized for solid content. 25 ml of the juice was evaporated to dryness in a tarred dish, cooled in a desiccator and weighed. Dosage of the juice used in the study is expressed in terms of solid content. The juice is reportedly non-toxic up to 10 g/kg (p.o) (Dhar et al., 1968; Mokkhasmit et al., 1971). 2.2. Estimation of total phenolics The total phenolics in the fresh juice of Cynodon dactylon shoots were determined by Folin’s–Ciocalteau method (Savitree et al., 2004). 90 l of fresh juice was made up to 1 ml (1 mg/ml) using methanol/water (20:30). To this, 5 ml of Folin’s–Ciocalteau phenol reagent (1:2), and 4 ml of 7.5% sodium carbonate were added and mixed well. After 90 min, the absorbance of the solution at 750 nm was measured with a double beam spectrophotometer (Perkin Elmer Lambda 25). Quantisation was based on the standard curve of gallic acid (0–100 g/ml) dissolved in methanol. 2.3. Effect of Cynodon dactylon on human DNA The DNA protective activity of fresh juice of Cynodon dactylon was determined according to Rajendra et al. (2006). 2.3.1. Pretreatment and isolation of DNA from human blood Venous blood containing ACD as an anticoagulant was obtained from the blood bank of Sri Ramachandra Medical College Hospital, Chennai. Blood samples, 5 ml each were withdrawn into 5 vials. Four of these samples were incubated with 50 g doxorubicin for 30 min. One sample which was untreated served as normal control. While one doxorubicin treated sample served as positive control, the others were treated with 3.26, 6.73 and 13.46 mg (equivalent to 50, 100
and 200 mg/kg body weight human dose) of Cynodon dactylon juice respectively. The samples were incubated for 1 h and 10 ml of red cell lysis buffer (10 mM Tris, 10 mM MgCl2 , 10 mM KCl and 2 mM EDTA, pH 8) and 1% Triton X-100, and incubated at 37 ◦ C for 5 min. The samples were centrifuged at 2000 rpm and the pellets were collected. They were resuspended in 10 ml of red cell lysis buffer and recentrifuged. The pellets were suspended in 1 ml of nucleated cell lysis buffer (10 mM Tris, 10 mM MgCl2 , 10 mM KCl, 2 mM EDTA and 400 mM NaCl, pH 8) and 0.2% sodium dodecyl sulphate, incubated at 55 ◦ C for 1 h, and centrifuged at 10,000 rpm for 10 min. DNA was precipitated by adding double the volume of ice cold ethanol to the supernatant. Strands of DNA were spooled out with an autoclaved tip, washed with 70% ethanol, and resuspended in 100 l of TE buffer (10 mM Tris and 1 M EDTA, pH 7.4) and set aside at 4 ◦ C for 12 h. The quality of DNA was assessed by wavelength scan spectra and agarose gel electrophoresis. 2.3.2. Wavelength scan spectra The purified DNA was diluted with distilled water (1:5) and scanned in the wavelength range of 200–400 nm using UV spectrophotometer against reagent blank. The absorbance of the samples at 260 and 280 nm was noted. 2.3.3. Agarose gel electrophoresis A 2% agarose solution in 1× TAE buffer (2 M Tris, 1N glacial acetic acid and 0.05 M EDTA, pH 8) with 4 l of ethidium bromide dispersed in it was poured in the gel rack. Upon solidification, it was placed in a tank containing 1× TAE buffer. Isolated DNA stained with 10 l of bromophenol blue, was injected into the wells formed before solidification, the lid was closed and 60 V current applied for 45 min. The tracks were visualized with UV light and photodocumented using BIORAD gel documentation system. 2.4. Immunomodulatory activity 2.4.1. Animals balb/c mice of either sex of average weight 25–30 g were used for the study approved by the Institutional Animal Ethics Committee. The experimental procedure was carried out in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The animals procured from the Central Animal Facility, Sri Ramachandra University were fed a standard rodent pellet diet and had free access to water. They were housed in polypropylene cages and maintained under standard laboratory conditions. 2.4.2. Humoral antibody response The effect of Cynodon dactylon on humoral antibody response was determined by the method adapted from Suresh et al. (1989). Groups of six mice each were immunized by injecting (i.p.) 0.2 ml SRBC (5 × 109 cells/ml) on day 0. They were divided into three groups and the fresh juice of Cynodon dactylon (equivalent to 250 and 500 mg/kg body weight) was administered orally on days −3, −2, −1, 0, 1, 2 and 3 to groups 2 and 3. Group 1 served as normal control and received no drug. On day 3 after immunization blood was collected from the retro orbital plexus and serum separated. Spleens were quickly removed, washed weighed and stored at 0 ◦ C until further use. 2.4.3. Haemagglutination antibody titer The standard microtechnique using 96-well microtiter plates was employed for the assay (Jaharapurkar et al., 2003). Twofold dilutions of sera were performed in 0.15 M phosphate buffered saline (pH 7.2), and 50 l of each dilution was aliquoted into each row of 96-well microtiter plates. 25 l of fresh 1% SRBC suspension in the phosphate buffered saline was dispersed into each well and
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mixed. The plates were incubated at 28 ◦ C for 2 h and examined visually for agglutination. The value of the highest serum dilution causing haemagglutination was taken as the antibody titer. 2.4.4. Plaque forming cell assay The technique of Jerne and Nordine (1963) was followed. The spleen cells were separated and collected in RPMI-1640 medium, washed twice and suspended in the same medium to a concentration of 106 cells/ml. Glass petri dishes were layered with 1.2% agarose in 0.15 M NaCl to form a bottom layer of agarose and incubated at 37 ◦ C for 90 min. A 2 ml quantity of 1:9 diluted fresh rabbit serum was added to each petri dish and further incubated for 4 min to allow the formation of plaques. Plaques were counted immediately using an inverted microscope (Nikon H-III power) and the values were expressed as counts per 106 spleen cells. 2.5. Statistical analyses All values are expressed as mean ± SEM. Statistical analyses were performed by Student’s t-test. Values of p lower than 0.05 were considered significant. 3. Results and discussion The fresh juice of Cynodon dactylon prepared in the manner indicated in traditional medicine was standardized for solid content (1.46%, w/w). The total phenolics in the juice were found to be 47 ± 0.33 mg/g gallic acid equivalents (GAE). The antioxidant action of polyphenols being well known, the total phenols in the herb could be mediating the immunomodulatory and DNA protective activity. The ability of the fresh juice of Cynodon dactylon to modulate doxorubicin-induced DNA damage has been studied by assessing the intactness of DNA isolated from human blood samples pretreated with the plant juice prior to doxorubicin incubation. DNA fragmentation pattern on agarose gel electrophoresis is shown in Fig. 1. The untreated control DNA in lane B shows no migration/fragmentation, an indication of its intact super coiled native form (Alessandria et al., 2003). A clear ladder formation is seen in lane A, evidence for breakage of native super coiled DNA into linear form and/or fragments as a result of treatment with the cytotoxic drug. There is no ladder formation in the lanes C, D and E showing intactness of DNA in samples treated with the fresh juice of Cynodon dactylon in the tested doses. Cytotoxicity of doxorubicin is attributed to its generation of free radicals (Silvina et al., 2003). The radical quenching antioxidant ability of the plant juice
Fig. 1. DNA fragmentation pattern in agarose gel electrophoresis. Lane A: doxorubicin (50 g) treated DNA; lane B: untreated native DNA; lane C: DNA pretreated with Cynodon dactylon juice (3.26 mg) before doxorubicin (50 g) treatment; lane D: DNA pretreated with Cynodon dactylon juice (6.73 mg) before doxorubicin (50 g) treatment; lane E: DNA pretreated with Cynodon dactylon juice (13.46 mg) before doxorubicin (50 g) treatment.
might explain its protective effect on DNA against doxorubicininduced damage. The wavelength scan spectra of DNA isolated from treated and untreated samples are shown in Fig. 2. The UV spectra of native DNA from the untreated control show a broad peak with a shoulder around 260 nm. Doxorubicin-treated DNA shows a flat spectrum with a change in wavelength of maximum absorbance indicating destabilization of configuration of DNA in the presence of doxorubicin. Spectra of DNA from samples treated with the plant juice show a broad peak with a shoulder similar to native DNA indicating minimum change in the DNA strands. While DNA from samples treated with the doses 3.26 and 6.73 mg show a similar and
Fig. 2. Ultraviolet spectral studies of DNA.
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Table 1 Effect of oral administration of fresh juice of Cynodon dactylon on antibody response in mice. S. no.
Treatment
Haemagglutination antibody titer
Plaque forming cell assay Weight of spleen (g)
1. 2. 3.
Normal control (no drug treatment) Fresh juice of Cynodon dactylon—250 mg/kg body weight Fresh juice of Cynodon dactylon—500 mg/kg body weight
3.0 ± 0.41 6.0 ± 0.82* 18.6 ± 14.40**
0.162 ± 0.010 0.182 ± 0.004 0.227 ± 0.025
Plaque forming cells (106 spleen cells) 510.50 ± 13.09 617.50 ± 24.57** 780.17 ± 16.20**
Each value is mean ± S.E.M. (n = 6). * p < 0.01 vs. control group. ** p < 0.005 vs. control group.
overlapping spectra, that of sample treated with 13.46 mg is of overall lesser absorbance. The ratio of absorbance of DNA at 260 and 280 nm for the untreated control is 1.48, while for the tested samples it is 1.66, 1.53 and 1.63, respectively, indicating nucleic acid purity in samples treated with Cynodon dactylon. For the positive control the ratio is 1.19, evidence of fragmentation (Glasel, 1995). Results of haemagglutination antibody titer and plaque forming cell assay are given in Table 1. There has been a dose-related increase in humoral antibody titer in the Cynodon dactylon treated groups and the rise is statistically significant. Therefore there has been an enhanced production of antibodies against antigen challenge when compared to untreated normal control. Also in the plaque forming cell assay fresh juice of Cynodon dactylon at both the tested doses showed an increase in the number of plaque forming cells compared to normal control. The increase has been dosedependent and the results are statistically significant. Also, there has been a non-significant increase in the weights of spleen of mice treated with Cynodon dactylon compared to normal control. The above results clearly demonstrate the immunostimulants nature of the fresh juice of Cynodon dactylon. In conclusion this study has clearly demonstrated the DNA protective activity and immunomodulatory property of the fresh juice of Cynodon dactylon. Its high phenolic content could explain the beneficial increase in the immune status of the animals. The proved immunomodulatory effect of the grass supports the reported antioxidant activity. The protective action of the plant against DNA damage gives scientific support for folklore claims of its usefulness as radio protective during eclipses. This study also validates the traditional use of the herb in ayurveda. Acknowledgements The authors would like to thank the technical support of the Department of Human Genetics, Sri Ramachandra University, Chennai, India. References Ahmed, S., Reza, M.S., Haider, S.S., Jabbar, A., 1994. Antimicrobial activity of Cynodon dactylon. Fitoterapia 65, 463–464. Alessandria, R., Angelo, A.I., Francesa, B., Marcella, R., Angelo, V., 2003. Free radical scavenging capacity and protectivity effect of Bacopa monniera L. on DNA damage. Phytotherapy Research 17, 870–875. Anonymous, 2001. The Wealth of India, A Dictionary of Indian Raw Materials and Industrial Products, First Supplement Series (Raw Materials), vol. 2. Council of Scientific and Industrial Research, New Delhi, pp. 331–332.
Artizzu, N., Bonsignore, L., Cottiglia, F., Loy, G., 1996. Studies on the diuretic and antimicrobial activity of Cynodon dactylon essential oil. Fitoterapia 67, 174–176. Auddy, B., Ferreira, M., Blasina, F., Lafon, L., Arredondo, F., Dajas, F., Tripathi, P.C., Seal, T., Mukherjee, B., 2003. Screening of antioxidant activity of 3 Indian medicinal plants, traditionally used for the management of neurogenerative diseases. Journal of Ethnopharmacology 84, 131–138. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary of Indian Medicinal Plants. Council for Scientific and Industrial Research, New Delhi, p. 88. Devasagayam, T.B.A., Saims, K.B., 2002. Immune system and antioxidants from Indian medicinal plants. Indian Journal of Experimental Biology 40, 639–655. Dhar, M.L., Dhar, M.M., Dhawan, B.N., Mehrotra, B.N., Ray, C., 1968. Screening of Indian medicinal plants for biological activity. Part I. Indian Journal of Experimental Biology 6, 232–247. Diasio, R.B., Lo Buglio, A.F., 1996. Immunomodulators: immunosuppressive agents and immunostimulants. In: Molinoft, P.B., Ruddon, R.W., Gilman, A.G. (Eds.), The Pharmacological Basis of Therapeutics. McGraw, New York, pp. 1291–1307. Govindarajan, R., Vijayakumar, M., Pushpangadan, P., 2005. Antioxidant approach to disease management and the role of ‘Rasayana’ herbs of Ayurveda. Journal of Ethnopharmacology 99, 165–178. Glasel, J.A., 1995. Validity of nucleic acid purities monitored by 260 nm/280 nm absorbance ratios. BioTechniques 18, 62–63. Jaharapurkar, S.P., Zambad, M.M., Wanjari, M.M., Umathe, S.N., 2003. In vivo evaluation of antioxidant activity of alcoholic extract of Rubia cordifolia Linn. and its influence on ethanol-induced immunosuppression. Indian Journal of Pharmacology 35, 232–236. Jerne, N.K., Nordine, A.A., 1963. Plaque forming agar by single antibody producing cells. Science 40, 405–407. Miller, R.P., 1967. Oestrogenic activity in Central African Highland grasses. Rhodesian Zambia Malawi Journal of Agricultural Research 5, 179. Mokkhasmit, M., Swatdimongkol, K., Satrawala, P., 1971. Study on toxicity of Thai medicinal plants. Bulletin of Department of Medical Sciences 12, 36–65. Nadkarni, A.K., 1976. Indian Materia Medica, vol. 1. Popular Prakashan, Bombay, p. 425. Ottmoto, T., Ikuse, M., Natori, S., 1970. Triterpenoids of the Graminae. Phytochemistry 9, 2137. Oudhia, P., 1999. Medicinal weeds in groundnut fields of Chattisghar (India). International Arachis Newsletter 19, 62–64. Rajendra, P., Sheetal, R.K., Sandeep, K., 2006. Hydrogen peroxide commences copper induced DNA damage isolated from human blood: in vitro study. Indian Journal of Experimental Biology 44, 377–380. Rizk, A.M., Heiba, H.I., Ma’Ayergi, H.A., Batanocing, K.H., 1986. Constituents of plants growing in Qatar. Fitoterapia 57, 3–9. Savitree, M., Isara, P., Nittaya, S., Worapan, S., 2004. Radical scavenging activity and total phenolic content of medicinal plants used in primary health care. SWU Journal of Pharmaceutical Sciences 9, 32–35. Silvina, B.N., Laura, M., Vargas, R., DarioCnello-Carrion, F., Daniel, R.C., 2003. Deoxyribonucleic acid damage induced by doxorubicin in peripheral blood mononuclear cells: possible role for the stress response and the deoxyribonucleic acid repair process. Cell Stress and Chaperones 4, 361–372. Subramanian, S., Nagarajan, S., 1988. Wound healing activity of Pongamia pinnata and Cynodon dactylon. Fitoterapia 59, 43–44. Suresh, K., Nirmala, K., Kezia, K., Vasudevan, D.M., 1989. Assessment of immunological parameters during tumour development in murine model. Indian Journal of Experimental Biology 27, 497–501. Thatte, U.M., Dahanukar, S.A., 1986. Ayurveda and contempory scientific thought. Trends in Pharmacological Sciences 7, 247–249.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Evaluation of the immunomodulatory and DNA protective activities of the shoots of Cynodon dactylon K. Mangathayaru a,∗ , M. Umadevi b , C. Umamaheswara Reddy a a Sri Ramachandra College of Pharmacy, Sri Ramachandra University, Porur, Chennai 600 116, India b RVS College of Pharmaceutical Sciences, Sulur, Coimbatore 641 402, India
a r t i c l e
i n f o
Article history: Received 28 June 2007 Received in revised form 29 September 2008 Accepted 13 February 2009 Available online 4 March 2009 Keywords: Cynodon dactylon Immunomodulatory DNA protective Rasayana drug
a b s t r a c t Ethnopharmacological relevance: Fresh juice of Cyanodon dactylon known as ‘durva’ grass is employed in India as a rejuvenator and for wound healing. Aim of the study: To validate the traditional use of the herb through evaluation of DNA protective activity in vitro and immunomodulatory activity in vivo. Materials and methods: Fresh juice of the grass was prepared as indicated for use in traditional medicine and standardized for solid content. Its total phenol content was estimated by Folin–Ciocalteau method. Freshly prepared juice was investigated for its effect on doxorubicin-induced DNA damage in vitro. Its immunomodulatory activity was tested on balb/c mice by the humoral antibody response which was determined by haemagglutination antibody titer and spleen cell assay. Results: Fresh juice of Cyanodon dactylon of 1.46% (w/w) solid content had a phenolic content of 47 ± 0.33 mg/kg GAE. At doses equivalent to 50, 100 and 200 mg total solids/kg body weight the juice protected human DNA against doxorubicin-induced DNA damage as demonstrated in DNA spectral studies, where the ratio of absorbance of DNA at 260 and 280 nm in samples pretreated with the juice was 1.66, 1.53 and 1.63 respectively, while it was 1.37 for DNA treated with doxorubicin only. This indicates nucleic acid purity in the Cynodon dactylon treated samples. Oral administration of the juice at 250 and 500 mg/kg in balb/c mice increased humoral antibody response upon antigen challenge, as evidenced by a dose-dependent, statistically significant increase in antibody titer in the haemagglutination antibody assay and plaque forming cell assay. Conclusions: The present report demonstrated the DNA protective activity and immunomodulatory property of the fresh juice of Cynodon dactylon validating the traditional use of the herb as a ‘rasayana’ in ayurvedic system of medicine. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The xenobiotic modulation of immune response is of current scientific interest due to its potential in the treatment of immunocompromised conditions (Thatte and Dahanukar, 1986). Currently available immunosuppressive and immunostimulating agents have major limitations, such as increased risk of infection and inhibition of cells formed by the bone marrow (Diasio and Lo Buglio, 1996).
Several ayurvedic medicinal plants are powerful immunomodulators (Devasagayam and Saims, 2002). Recent research has linked their therapeutic actions to their strong antioxidant potential (Govindarajan et al., 2005). Despite their therapeutic importance, very few of them have been investigated in detail. The objective of this work is to evaluate a standardized fresh juice of Cynodon dactylon with respect to DNA protective as well as immunomodulatory effect in view of the use of the herb in neurodegenerative and immunological disorders. 1.1. Plant
Abbreviations: ACD, acid–citrate–dextrose; EDTA, ethylenediaminetetraacetic acid; TE, tris–ethylenediaminetetraacetic acid; TAE, tris–acetic acid–ethylenediaminetetraacetic acid; SRBC, sheep red blood cells; GAE, gallic acid equivalent; DNA, deoxyribonucleic acid; HPTLC, high performance thin layer chromatography; NaCl, sodium chloride; KCl, potassium chloride; MgCl2 , magnesium chloride. ∗ Corresponding author. Tel.: +91 24768403x8930. E-mail address: [email protected] (K. Mangathayaru). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.02.036
Cynodon dactylon Linn. Pers. (Poaceae) was collected from in and around Chennai and authenticated by Prof. P. Jayaraman, Director of Plant Anatomy Research Centre, Chennai, India. A voucher specimen (COPG-PG-A13) was deposited in the herbarium of the Department of Pharmacognosy, Sri Ramachandra College of Pharmacy, Sri Ramachandra University.
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1.2. Uses in traditional medicine A hardy perennial commonly called Bermuda grass, Cynodon dactylon is one of the most commonly occurring weeds in India. A ‘Rasayana’ drug, it is an inseparable part of religious rituals and is a valuable herbal medicine used as first aid in minor injuries (Oudhia, 1999). In Indian households it is a common practice to place strands of this grass on eatables during eclipse. Folk wisdom claims it to be water purifier (Artizzu et al., 1996). The juice of the plant is astringent and the fresh juice is used in the treatment of chronic diarrhea and dysentery (The Wealth of India, 2001). The plant occupies a renowned position in ayurveda, unani and homoeopathic systems of medicine (Chopra et al., 1956). Wound healing (Subramanian and Nagarajan, 1988), antimicrobial (Ahmed et al., 1994), antioxidant (Auddy et al., 2003) and estrogenic (Miller, 1967) activities of the plant are earlier reported. 1.3. Previously isolated classes of constituents Compounds including arundoin, furfural, furfuralcohol, ionone, 2-(4 -hydroxyphenyl)-propionic, 4-hydroxybenzoic, 2-(3 methoxy-4-hydroxyphenyl) propionic, and 3-methoxy-4-hydroxybenzoic acids, phytol, -sitosterol-d-glucoside, stigmasterol acetate and phytone have been isolated from Cynodon dactylon (Miller, 1967; Ottmoto et al., 1970; Rizk et al., 1986; Artizzu et al., 1996). 2. Materials and methods 2.1. Preparation of fresh juice The fresh shoots of Cynodon dactylon (1 kg) were washed, airdried and ground in a food processor and filtered. The juice so prepared as indicated for use in traditional medicine (Nadkarni, 1976) was standardized for solid content. 25 ml of the juice was evaporated to dryness in a tarred dish, cooled in a desiccator and weighed. Dosage of the juice used in the study is expressed in terms of solid content. The juice is reportedly non-toxic up to 10 g/kg (p.o) (Dhar et al., 1968; Mokkhasmit et al., 1971). 2.2. Estimation of total phenolics The total phenolics in the fresh juice of Cynodon dactylon shoots were determined by Folin’s–Ciocalteau method (Savitree et al., 2004). 90 l of fresh juice was made up to 1 ml (1 mg/ml) using methanol/water (20:30). To this, 5 ml of Folin’s–Ciocalteau phenol reagent (1:2), and 4 ml of 7.5% sodium carbonate were added and mixed well. After 90 min, the absorbance of the solution at 750 nm was measured with a double beam spectrophotometer (Perkin Elmer Lambda 25). Quantisation was based on the standard curve of gallic acid (0–100 g/ml) dissolved in methanol. 2.3. Effect of Cynodon dactylon on human DNA The DNA protective activity of fresh juice of Cynodon dactylon was determined according to Rajendra et al. (2006). 2.3.1. Pretreatment and isolation of DNA from human blood Venous blood containing ACD as an anticoagulant was obtained from the blood bank of Sri Ramachandra Medical College Hospital, Chennai. Blood samples, 5 ml each were withdrawn into 5 vials. Four of these samples were incubated with 50 g doxorubicin for 30 min. One sample which was untreated served as normal control. While one doxorubicin treated sample served as positive control, the others were treated with 3.26, 6.73 and 13.46 mg (equivalent to 50, 100
and 200 mg/kg body weight human dose) of Cynodon dactylon juice respectively. The samples were incubated for 1 h and 10 ml of red cell lysis buffer (10 mM Tris, 10 mM MgCl2 , 10 mM KCl and 2 mM EDTA, pH 8) and 1% Triton X-100, and incubated at 37 ◦ C for 5 min. The samples were centrifuged at 2000 rpm and the pellets were collected. They were resuspended in 10 ml of red cell lysis buffer and recentrifuged. The pellets were suspended in 1 ml of nucleated cell lysis buffer (10 mM Tris, 10 mM MgCl2 , 10 mM KCl, 2 mM EDTA and 400 mM NaCl, pH 8) and 0.2% sodium dodecyl sulphate, incubated at 55 ◦ C for 1 h, and centrifuged at 10,000 rpm for 10 min. DNA was precipitated by adding double the volume of ice cold ethanol to the supernatant. Strands of DNA were spooled out with an autoclaved tip, washed with 70% ethanol, and resuspended in 100 l of TE buffer (10 mM Tris and 1 M EDTA, pH 7.4) and set aside at 4 ◦ C for 12 h. The quality of DNA was assessed by wavelength scan spectra and agarose gel electrophoresis. 2.3.2. Wavelength scan spectra The purified DNA was diluted with distilled water (1:5) and scanned in the wavelength range of 200–400 nm using UV spectrophotometer against reagent blank. The absorbance of the samples at 260 and 280 nm was noted. 2.3.3. Agarose gel electrophoresis A 2% agarose solution in 1× TAE buffer (2 M Tris, 1N glacial acetic acid and 0.05 M EDTA, pH 8) with 4 l of ethidium bromide dispersed in it was poured in the gel rack. Upon solidification, it was placed in a tank containing 1× TAE buffer. Isolated DNA stained with 10 l of bromophenol blue, was injected into the wells formed before solidification, the lid was closed and 60 V current applied for 45 min. The tracks were visualized with UV light and photodocumented using BIORAD gel documentation system. 2.4. Immunomodulatory activity 2.4.1. Animals balb/c mice of either sex of average weight 25–30 g were used for the study approved by the Institutional Animal Ethics Committee. The experimental procedure was carried out in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The animals procured from the Central Animal Facility, Sri Ramachandra University were fed a standard rodent pellet diet and had free access to water. They were housed in polypropylene cages and maintained under standard laboratory conditions. 2.4.2. Humoral antibody response The effect of Cynodon dactylon on humoral antibody response was determined by the method adapted from Suresh et al. (1989). Groups of six mice each were immunized by injecting (i.p.) 0.2 ml SRBC (5 × 109 cells/ml) on day 0. They were divided into three groups and the fresh juice of Cynodon dactylon (equivalent to 250 and 500 mg/kg body weight) was administered orally on days −3, −2, −1, 0, 1, 2 and 3 to groups 2 and 3. Group 1 served as normal control and received no drug. On day 3 after immunization blood was collected from the retro orbital plexus and serum separated. Spleens were quickly removed, washed weighed and stored at 0 ◦ C until further use. 2.4.3. Haemagglutination antibody titer The standard microtechnique using 96-well microtiter plates was employed for the assay (Jaharapurkar et al., 2003). Twofold dilutions of sera were performed in 0.15 M phosphate buffered saline (pH 7.2), and 50 l of each dilution was aliquoted into each row of 96-well microtiter plates. 25 l of fresh 1% SRBC suspension in the phosphate buffered saline was dispersed into each well and
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mixed. The plates were incubated at 28 ◦ C for 2 h and examined visually for agglutination. The value of the highest serum dilution causing haemagglutination was taken as the antibody titer. 2.4.4. Plaque forming cell assay The technique of Jerne and Nordine (1963) was followed. The spleen cells were separated and collected in RPMI-1640 medium, washed twice and suspended in the same medium to a concentration of 106 cells/ml. Glass petri dishes were layered with 1.2% agarose in 0.15 M NaCl to form a bottom layer of agarose and incubated at 37 ◦ C for 90 min. A 2 ml quantity of 1:9 diluted fresh rabbit serum was added to each petri dish and further incubated for 4 min to allow the formation of plaques. Plaques were counted immediately using an inverted microscope (Nikon H-III power) and the values were expressed as counts per 106 spleen cells. 2.5. Statistical analyses All values are expressed as mean ± SEM. Statistical analyses were performed by Student’s t-test. Values of p lower than 0.05 were considered significant. 3. Results and discussion The fresh juice of Cynodon dactylon prepared in the manner indicated in traditional medicine was standardized for solid content (1.46%, w/w). The total phenolics in the juice were found to be 47 ± 0.33 mg/g gallic acid equivalents (GAE). The antioxidant action of polyphenols being well known, the total phenols in the herb could be mediating the immunomodulatory and DNA protective activity. The ability of the fresh juice of Cynodon dactylon to modulate doxorubicin-induced DNA damage has been studied by assessing the intactness of DNA isolated from human blood samples pretreated with the plant juice prior to doxorubicin incubation. DNA fragmentation pattern on agarose gel electrophoresis is shown in Fig. 1. The untreated control DNA in lane B shows no migration/fragmentation, an indication of its intact super coiled native form (Alessandria et al., 2003). A clear ladder formation is seen in lane A, evidence for breakage of native super coiled DNA into linear form and/or fragments as a result of treatment with the cytotoxic drug. There is no ladder formation in the lanes C, D and E showing intactness of DNA in samples treated with the fresh juice of Cynodon dactylon in the tested doses. Cytotoxicity of doxorubicin is attributed to its generation of free radicals (Silvina et al., 2003). The radical quenching antioxidant ability of the plant juice
Fig. 1. DNA fragmentation pattern in agarose gel electrophoresis. Lane A: doxorubicin (50 g) treated DNA; lane B: untreated native DNA; lane C: DNA pretreated with Cynodon dactylon juice (3.26 mg) before doxorubicin (50 g) treatment; lane D: DNA pretreated with Cynodon dactylon juice (6.73 mg) before doxorubicin (50 g) treatment; lane E: DNA pretreated with Cynodon dactylon juice (13.46 mg) before doxorubicin (50 g) treatment.
might explain its protective effect on DNA against doxorubicininduced damage. The wavelength scan spectra of DNA isolated from treated and untreated samples are shown in Fig. 2. The UV spectra of native DNA from the untreated control show a broad peak with a shoulder around 260 nm. Doxorubicin-treated DNA shows a flat spectrum with a change in wavelength of maximum absorbance indicating destabilization of configuration of DNA in the presence of doxorubicin. Spectra of DNA from samples treated with the plant juice show a broad peak with a shoulder similar to native DNA indicating minimum change in the DNA strands. While DNA from samples treated with the doses 3.26 and 6.73 mg show a similar and
Fig. 2. Ultraviolet spectral studies of DNA.
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Table 1 Effect of oral administration of fresh juice of Cynodon dactylon on antibody response in mice. S. no.
Treatment
Haemagglutination antibody titer
Plaque forming cell assay Weight of spleen (g)
1. 2. 3.
Normal control (no drug treatment) Fresh juice of Cynodon dactylon—250 mg/kg body weight Fresh juice of Cynodon dactylon—500 mg/kg body weight
3.0 ± 0.41 6.0 ± 0.82* 18.6 ± 14.40**
0.162 ± 0.010 0.182 ± 0.004 0.227 ± 0.025
Plaque forming cells (106 spleen cells) 510.50 ± 13.09 617.50 ± 24.57** 780.17 ± 16.20**
Each value is mean ± S.E.M. (n = 6). * p < 0.01 vs. control group. ** p < 0.005 vs. control group.
overlapping spectra, that of sample treated with 13.46 mg is of overall lesser absorbance. The ratio of absorbance of DNA at 260 and 280 nm for the untreated control is 1.48, while for the tested samples it is 1.66, 1.53 and 1.63, respectively, indicating nucleic acid purity in samples treated with Cynodon dactylon. For the positive control the ratio is 1.19, evidence of fragmentation (Glasel, 1995). Results of haemagglutination antibody titer and plaque forming cell assay are given in Table 1. There has been a dose-related increase in humoral antibody titer in the Cynodon dactylon treated groups and the rise is statistically significant. Therefore there has been an enhanced production of antibodies against antigen challenge when compared to untreated normal control. Also in the plaque forming cell assay fresh juice of Cynodon dactylon at both the tested doses showed an increase in the number of plaque forming cells compared to normal control. The increase has been dosedependent and the results are statistically significant. Also, there has been a non-significant increase in the weights of spleen of mice treated with Cynodon dactylon compared to normal control. The above results clearly demonstrate the immunostimulants nature of the fresh juice of Cynodon dactylon. In conclusion this study has clearly demonstrated the DNA protective activity and immunomodulatory property of the fresh juice of Cynodon dactylon. Its high phenolic content could explain the beneficial increase in the immune status of the animals. The proved immunomodulatory effect of the grass supports the reported antioxidant activity. The protective action of the plant against DNA damage gives scientific support for folklore claims of its usefulness as radio protective during eclipses. This study also validates the traditional use of the herb in ayurveda. Acknowledgements The authors would like to thank the technical support of the Department of Human Genetics, Sri Ramachandra University, Chennai, India. References Ahmed, S., Reza, M.S., Haider, S.S., Jabbar, A., 1994. Antimicrobial activity of Cynodon dactylon. Fitoterapia 65, 463–464. Alessandria, R., Angelo, A.I., Francesa, B., Marcella, R., Angelo, V., 2003. Free radical scavenging capacity and protectivity effect of Bacopa monniera L. on DNA damage. Phytotherapy Research 17, 870–875. Anonymous, 2001. The Wealth of India, A Dictionary of Indian Raw Materials and Industrial Products, First Supplement Series (Raw Materials), vol. 2. Council of Scientific and Industrial Research, New Delhi, pp. 331–332.
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