**4. Results and discussion**

The compound isolated from *G. lucidum* answered anthrone and phenol sulphuric tests giving typical colour reactions indicating the presence of carbohydrates. From the IR spectrum, pyranoid form was suggested to be present due to the presence of three absorption bands at 1153.4, 1091.6 and 1029.9 cm<sup>−</sup><sup>1</sup> . In the HNMR spectrum H<sup>−</sup><sup>1</sup> signals were observed at less than 4.8 ppm (4.762, 4.683, 4.667, 4.658, 4.402 ppm), which suggest that component sugars have beta configuration. From gel filtration chromatography, the molecular weight of the compound was found to be 1.5 × 106 Daltons. From the acid hydrolysis treatment for the detection of monosaccharides, the sugars present in the compound were found to be glucose, mannose and rhamnose. The compound was identified to be beta-glucan.

#### **4.1 DNA repair enhancement**

The repair process in lymphocytes was found to be enhanced by the glucan at 50 μg/ml concentration. The percent DNA, tail length, tail moment and olive tail moment was reduced significantly. At 2 Gy 0 min, the comet parameters increased. Fifteen minutes after irradiation the comet parameters were reduced. The presence of glucan reduced the comet parameters further. After 2 h of irradiation the comet parameters were reduced by the glucan to the control level (**Figure 4** and **Table 1**).

#### **Figure 4.**

*DNA repair enhancement by glucan in human lymphocytes (comet assay). Untreated: (a) control; (c) 2 Gy 0 min; (e) 2 Gy 15 min; (g) 2 Gy 30 min; (i) 2 Gy 45 min; (k) 2 Gy 60 min; (m) 2 Gy 120 min. Treated with glucan: (b) control; (d) 2 Gy 0 min; (f) 2 Gy 15 min; (h) 2 Gy 30 min; (j)2 Gy 45 min; (l) 2 Gy 60 min; (n) 2 Gy 120 min.* 


*Datas are mean ± S.E. n = 6. a P < 0.0001. b P < 0.001. c P < 0.01. d P < 0.05. e Marginally significant, compared to RT alone. f P < 0.05 compared to RT + amifostine. g P < 0.0001 compared to DDW. h P < 0.001 compared to amifostine alone. i P < 0.01 compared to amifostine alone. j P < 0.05 compared to amifostine alone. k P < 0.0001 compared to glucan alone. l P < 0.001 compared to glucan alone.* 

#### **Table 1.**

*Effect of G. lucidum glucan and amifostine on the induction of different chromosomal aberrations in mouse bone marrow after whole body γ-irradiation (4 Gy).* 

#### **4.2 Chromosomal aberrations**

 Sham treated control showed 1% aberrant cells. Compared to control glucan or amifostine alone did not induce any significant changes. There was significant increase in the percentage of aberrant cells treated with radiation. Treatment with glucan after irradiation and amifostine before irradiation resulted in significant

#### **Figure 5.**

*Different types of chromosomal aberrations in mouse bone marrow. F, fragments; CD, chromatid break; CB, chromosome break; PN, pulverisation; SDC, severe damaged cell; PP, polyploidy; D, dicentrics; R, rings.* 

 decrease in the percentage of aberrant cells and number of aberrations per cell compared to the group which received radiation alone. A decrease in all types of aberrations, as well as polyploidy and cells with pulverisation was observed. The number of severe damaged cells (SDC) significantly reduced to about 1.5 times after glucan treatment. The number of cells with multiple and complex damage was


*Datas are mean ± S.E. n = 6.* 

*a P < 0.0001. b P < 0.001. c P < 0.01. d P < 0.05. e Marginally significant, compared to RT alone. f P < 0.05 compared to RT + amifostine. g P < 0.0001 compared to DDW. h P < 0.001 compared to amifostine alone. i P < 0.01 compared to amifostine alone. j P < 0.05 compared to amifostine alone. k P < 0.0001 compared to glucan alone. l P < 0.001 compared to glucan alone.* 

#### **Table 2.**

*Effect of G. lucidum polysaccharides and amifostine on the induction of polyploidy, SDC and pulverization in mouse bone marrow after whole body γ-irradiation (4 Gy).* 


#### **Table 3.**

*Effect of glucan on enhancement of DNA repair in human lymphocytes after 2 Gy gamma irradiation (comet assay).* 

significantly decreased by glucan post-treatment indicating that the former may help in the repair of the DNA breaks (**Figure 5**, **Tables 2** and **3**).

The lifespan of cells to radiation leading to a loss of cell viability can be greatly influenced by the ability of cells to repair injured DNA. The hazard in mammals exposed to ionizing radiation is to the haemopoetic system. Radiation induced damage to DNA can temporarily affect DNA replication allowing repair to happen involving a well-coordinated event of DNA repair enzymes such as DNA repair polymerase, DNA ligase and PARP [28]. The factors that influence the response of living cells to radiation are the DNA repair status, the physiological state of cells, the presence of oxygen and chemicals as well as pre and post-irradiation treatments [29].

 By examining the comet parameters of human peripheral blood leucocytes the effect of polysaccharides on DNA repair was ascertained. Through the initial 30 min, most of the DNA repair processes were completed. The presence of polysaccharide boosted the process of DNA repair. The comet parameters were more at 30 min post-irradiation, in irradiated control and polysaccharide treated group which can be attributed to the commencement of excision repair process [30]. After 45 min

 there was not much difference in the comet parameters, in control group. The comet parameters kept on reducing in the presence of polysaccharides and at 120 min the comet parameters were almost similar to the unirradiated control. Re-joining of DNA strand breaks by most cell types is known to be a rapid process within few seconds-minutes [31] and this kinetics are seen in comet assay too. In freshly isolated lymphocytes repair by Hydrogen peroxide induced breaks takes place very slowly which can be due to the additional DNA breakage as a result of quick exposure to atmospheric oxygen in the repair incubation period [32]. At the same time repair of endonuclease III- or FPG-sensitive sites (i.e., oxidized purine and pyrimidines) by base excision repair, is much slower process, taking few hours [33].

Background levels of DNA damage in normal cells, the variation in DNA repair capacity within human populations, and the regulation of DNA repair at the molecular level within the nucleus can be monitored by comet assay [34].

#### **5. Conclusion**

The integrity of DNA molecule at structural level has to be protected and preserved for the effectual transmission of the genetic information contained to progeny. Distinctions in the arrangement of nucleotides or changes in the configuration of bases or sugars, in the double helix of DNA can impede the replication or transcription of genome.

Multilation to DNA molecule is the crucial factor for cell death. Mechanisms of repair of damaged DNA molecules play a vital role in cell survival. No medicine has been invented that could successively be applied in DNA damage. Our study indicates that the polysaccharides from *G. lucidum* enhance the repair process.

#### **5.1 Advances in area of DNA repair**

 Prevention is better than cure and cancer induction is greatly influenced by nutrition. The unaffordable discovery cost and failures at the completion of discovery pipeline makes medicines arbitrary to the developing countries. Newer technologies like reverse pharmacology, systems biology which are charming give innovation opportunities based on investigational wisdom and universal viewpoint of translation medicine. Chemotherapy and SSRI revolutionised longevity and quality of life in therapeutics. The Human Genome Project opened understanding towards personalised medicine. Glucan from *G. lucidum* possess immunomodulating activities and regulate a number of undiscovered cellular genes. New studies are needed to unravel these molecular targets giving insights into the interactions of the fungi like *G. lucidum* with our body system and provide strategies for the discovery of effective and safe approaches for drugs from natural sources.

Glucan was isolated from the mushroom *Ganoderma lucidum*, a basidiomycete white rot macro fungus that has been used extensively for therapeutic use in China and Japan for years. The compound was characterised by different chromatographic techniques, done by IR, NMR, and paper chromatography, gel filtration chromatography and spectroscopic techniques like infra-red spectrum and nuclear magnetic resonance spectrum.

The molecular weight of the isolated glucan was 1.6 × 106 Daltons. The rate of DNA repair in the presence and absence of the compound was determined. Comet assay was performed using the method of Singh in human lymphocytes. Chromosomal aberration was studied in mouse bone marrow. After radiation exposure, the comet parameters, percent DNA, tail length, tail moment and olive tail moment were changed in the presence of glucan. Chromosomal aberrations and

#### *Natural Drugs in DNA Repair DOI: http://dx.doi.org/10.5772/intechopen.86008*

individual aberrations were also reduced by glucan. The result of present investigation reveals the potential application of glucan from *G. lucidum* in increasing the rate of DNA repair which makes it useful in medical scenario.

 The path of science is always fascinating giving deep intuitions with new technologies. The term 'DNA repair' gained more significance in last decade. The beautiful discoveries in essential mechanisms of DNA repair extended Nobel prize in Chemistry in 2015 to T. Lindahl, P. Modrich and A. Sancar. Their discovery defined three pathways that essentially correct DNA damage, protecting the integrity of genetic code assuring perfect replication through generations allowing correct cell division. The mechanisms behind base excision repair, mismatch repair and nucleotide excision repair was explained. Since then the number of drugs and targeted pathways has increased remarkably. The DNA repair enzyme was declared as the molecule of the year in 1994. Though the studies from model organisms serve as a basis to elucidate of repair mechanism, the utilisation of cutting edge technology has channelled in a new era of DNA repair research. The DNA repair pathways have also become better understood. The accessibility of a wide-ranging spectrum of drugs with known molecular targets will provide the rationale to use those drugs in relation to various disease conditions and to combine DNA damaging agents with the appropriate DNA repairing agent. The journey of DNA repair continues. Our current research is carried out in this direction.
