A PHARMACEUTICAL COMPOSITION TO INHIBIT CHROMOSOMAL DAMAGE

Abstract

A PHARMACEUTICAL COMPOSITION TO INHIBIT CHROMOSOMAL DAMAGE A pharmaceutical composition to inhibit chromosomal damage relates to the pharmaceutical composition comprising Desmostachya bipinnata extract as the key ingredient for inhibiting damage to chromosomes and the method of preparation thereof. The results are as par with the previously reported an radioprotective activity by various authors such as, Ayumi Yamamato et.al., reported the radioprotective activity of blackcurrant extract evaluated by in-vitro micronucleus and gene mutation assays in TK6 human lymphoblatoid cells.

Specification

Description:A PHARMACEUTICAL COMPOSITION TO INHIBIT CHROMOSOMAL DAMAGE

FIELD OF INVENTION
The invention relates to a pharmaceutical composition for inhibiting damage to chromosomes, more particularly related to a Desmostachya bipinnata extract for inhibiting damage to chromosomes and method for the preparation thereof.
BACKGROUND OF THE INVENTION
Genotoxicity is the potential of a substance/agent to have a serious impact on Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA), thus affecting their consistency. Today, the Global System for Mobile Telecommunications mobile phone, RF radiation of frequencies ranging around 880 and 1,800 MHz, is considered one of the most widely used RF transmitters in our daily life. They have adverse effects on public health. Fast-growing wireless broadband and networking systems have been the primary driving force of the emission levels, posing risks to the atmosphere and human life while also offering tangible alternatives to globalization's changing needs. Desmostachya bipinnata was found to show effective DNA protective action in-vitro.
Waffa A Mohamed et.al., reported ameliorative effect of spirulina platensis against radiation-induced genotoxicity in male Sprague Dawley Rats. The results were observed through % tail length through comet assay and MPCE's from bone marrow cells. The study reported that the genotoxic effect of GSM 900MHz could attributed to the free radical especially OH generated in the tissues via RF particles that could lead to oxidative damage to the DNA. Author concluded that long time exposure to MR alters blood picture and has degenerative effects on bone marrow cells.
Reza et.al., reported the genoprotective effects of the grape seed extract against Gamma radiation induced genotoxicity in bone marrow cells of mice. The study gave a brief description on the genoprotective effect of the GSE at the dose 200mg/kg/bw which could reduce the cytotoxicity and clastogenicity. The author concluded the GSE was shown genoprotective and radioprotective action by the antioxidant and free radical scavenging property of the Grape seed extract.
Though the prior arts research on the same subject matter, there is no research activities reported on genoprotective nature of Desmostachya Bipinnata. In the present invention the extract of Desmostachya Bipinnata, is found to show effective DNA protective action in-vitro with the evidences of % tail moment and % MN frequency.
Therefore it is object of the invention to provide a pharmaceutical composition for inhibiting damage to chromosomes.
Another object of the invention to provide to a Desmostachya bipinnata extract for inhibiting damage to chromosomes.
Further object of the invention to provide a method for the extraction of Desmostachya bipinnata using solvents.

SUMMARY OF THE INVENTION
It is therefore the present invention provides pharmaceutical composition comprising Desmostachya bipinnata extract for inhibiting damage to chromosomes.
Another aspect of the invention to provide a pharmaceutical composition for inhibiting damage to chromosomes comprising; Desmostachya bipinnata extract - 0.5-80%; diluents from 0% to 78.0%; lubricants from 0% to 3.0%; binders from 0% to 5.0%; and other pharmaceutically acceptable excipients.
Still another aspect of the invention to provide a method for preparing the Desmostachya Bipinnata extract powder comprising; drying the whole plant Desmostachya bipinnata in a shade for about 10 days; pulverizing into coarse powder; extracting the active material by water extraction process and kept in a shaking incubator for 2- 3hrs at 200rpm and filtering the extract and evaporated to dry powder.
BRIEF DESCRIPTION OF DRAWINGS
The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

Fig. 1 illustrates the DNA damage and formation of the olive and tail movement of the bone marrow cells of the RFR induction. Images explaining both pre & post treated groups with 100mg/kg & 200mg/kg respectively. Fig. 2 illustrates the results of the % olive movement of the G1 & other treated groups. Fig. 3 illustrates the results of the % tail movement/ tail length Fig. 4 illustrates the effect of the Desmostachya bipinnata extract treated groups against the RFR exposed group
Other aspects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION
The following descriptions are exemplary embodiments only, and are not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention as set forth in the appended claims.
Moreover, all statements herein reciting embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents as well as equivalents developed in the future (i.e., any elements developed that perform the same function, regardless of structure).
As used herein, the term "comprising" means that other steps and other components that do not affect the end result may be utilized. The term "comprising" encompasses the expressions "consisting of "and "consisting essentially of". The use of singular identifiers such as "the", "a", or "an" is not intended to be limiting solely to the use of a single component, but may include multiple components.
An oral dosage form wherein a mixture of an active compound comprising Desmostachya bipinnata extract powder mixed with one or more auxiliary agent, and further the blends are tableted or enclosed in capsule. The excipients are selected for the best dissolution and releases the active ingredient from the dosage form as fast as possible.
The term "active ingredient" refers to an agent, active ingredient compound or other substance, or compositions and mixture thereof that provide some pharmacological, often beneficial, effect. Reference to a specific active ingredient shall include where appropriate the active ingredient is Desmostachya bipinnata extract powder and it's pharmaceutically acceptable salts.
The term "dosage form" denotes any form of the formulation that contains an amount sufficient to achieve a therapeutic effect.
A pharmaceutical dosage form of a tablet or capsule or parenteral which comprises a pharmaceutically effective amount of active ingredient containing Desmostachya bipinnata extract powder and other excipients.
The present invention also provides a process for preparation of solid dosage form preferably tablet or capsule of Desmostachya bipinnata extract with one or more adjuvants.

Pharmaceutical composition according to the present invention is preferably in the form of a tablet or capsule or parenteral. The active ingredient used in the present pharmaceutical composition is Desmostachya bipinnata extract.
The solid dosage form tablet or capsule of the present invention is prepared using active ingredient and pharmaceutically acceptable excipients selected from the group comprising of diluents, lubricants, glidants and other pharmaceutically acceptable excipients or adjuvants.
Diluents may be selected from the group comprising of mannitol, microcrystalline Cellulose, lactose, starch, dibasic calcium phosphate anhydrous, tribasic calcium phosphate, kaolin, sucrose, precipitated calcium carbonate, sorbitol, maltodextrin, powdered cellulose, micro crystalline cellulose and other materials known for such property. Diluents in the dosage form ranges from 0% to 78.0% by weight.
Lubricants may be selected from the group comprising of stearic acid, sodium stearyl fumarate, polyethylene glycol, magnesium stearate, calcium stearate, talc, zinc stearate, hydrogenated castor oil, silica, colloidal silica, cornstarch, calcium silicate, magnesium silicate, silicon hydrogel and other materials known for such property. Lubricants in the dosage form ranges from 0% to 3.0% by weight.
Binders may be selected from the group comprising of polyvinylpyrrolidone, hydroxypropyl methylcellulose, acacia, alginic acid, hydroxy propyl cellulose, carboxymethylcellulose sodium, compressible sugar, ethylcellulose, gelatin, liquid glucose, methylcellulose, pregelatinized starch and other materials known to one of ordinary skill in the art. Binders in the dosage form ranges from 0% to 5.0% by weight.
Glidants may be selected from the group comprising of colloidal silicon dioxide, colloidal silica, cornstarch, talc, calcium silicate, magnesium silicate, colloidal silicon, silicon hydrogel and other materials known for such property. Glidants in the dosage form ranges from 0% to 2.0% by weight.
The pharmaceutical composition may optionally be coated with functional and/or non-functional layers comprising film-forming polymers, if desired. Examples of film-forming polymers include ethylcellulose, hydroxypropyl methylcellulose, hydroxypropylcellulose, polyvinyl alcohol, polyvinyl acetate methylcellulose, carboxymethyl cellulose, hydroxymethylcellulose, hydroxyethylcellulose, cellulose acetate, hydroxypropyl methylcellulose phthalate, cellulose acetate phthalate, cellulose acetate trimellitate; waxes; methacrylic acid polymers and the like. Alternatively, commercially available coating compositions comprising film-forming polymers marketed under various trade names, such as Opadry may also be used for coating. Film-forming polymer in the dosage form ranges from 1.5% to 4.0% by weight.
The alkalising agent(s), which are to be used in accordance with the invention, are now described. It is only required of such alkalising agents to show basicity (pH of not less than 7) when they are in the form of a 1% aqueous solution or suspension alkalising agent may be selected from the group comprising sodium dihydrogen phosphate disodium dihydrogen phosphate, trisodium phosphate, sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, potassium hydrogen phosphate, dipotassium hydrogen phosphate, tripotassium phosphate and other materials known for such property. Applicants also found certain alkalising agent is more efficient in releasing active ingredient from the oral dosage form than other common dissolution improving agents, such as sodium lauryl sulphate, polysorbate, starch, mannitol, lactose, pre-gelatinised starch, microcrystalline cellulose.
One skilled in the art would recognize other suitable auxiliary agents, lubricants and glidants that can be used in the composition of present invention.

The invention will be further clarified by the following examples, which are intended to be purely exemplary of the invention and are not intended to limit the scope of the invention.
Example 1:
Composition 1
Desmostachya bipinnata extract - 100mg
mannitol - 151mg
microcrystalline cellulose - 65mg
sodium Stearyl Fumarate - 5mg

Example 2:
Composition 2
Desmostachya bipinnata extract - 200mg
mannitol - 302mg
microcrystalline cellulose - 130mg
sodium Stearyl Fumarate - 10mg
hydroxy propyl methylcellulose - 13mg
polyethylene glycol 6000 - 3mg

Example 3:
Method for preparing the Desmostachya bipinnata extract powder:
The whole plant Desmostachya Bipinnata was shade-dried for about 10 days and the dried plant was pulverized into coarse powder. The dried powder was subjected to an extraction process using water as a solvent. The aqueous extract of the dried whole plant powder was made in the distilled water. The mixture was then taken into 250ml sterile conical flasks, plugged with sterile cotton, and kept in a shaking incubator for about 3hrs at 200rpm in a room temperature. The extract was filtered and evaporated to obtain dry powder.
Example 4:
Desmostachya bipinnata extract with suitable excipients are sifted through suitable sieve and optionally with suitable excipients and granulate with suitable solvents. The wet granules were dried at suitable temperature and size through suitable sieve. The dried granules were blended with lubricant and optionally with diluents. Filling this blend into capsule or compress the lubricated blend into tablets.
Example 5
Genotoxicity study:
A wireless XYZ mobile phone of GSM-900MHz frequency range was used. This served as an induction model for the study. The phone was completely charged. After this, it was placed in the middle of the cages. It was positioned at a distance of 10cm above the animal cages. The animals were exposed to the radiation field of mobile phone. The phone was kept in non-speaking mode. Two mobile phones which were of the same frequency (GSM-900MHz) range were used. Throughout the study control and treated groups received specific intervals of radiation.
After the acclimatization for 5 days the study was started on the 6th day and animals were subjected to randomization and were grouped according to their body weight. The groups were served as Normal (G1), for which the portable water 10ml/kg/bw was given, Control group (G2) which was exposed to the MR modulations of 900MHz, Pre-treatment (G3,G4)groups of both LD and HD were given 100mg/kg/bw and 200mg/kg/bw of Desmostachya bipinnata extract powder respectively, Post-treatment (G5,G6) groups of both LD and HD of 100 and 200mg/kg/bw Desmostachya bipinnata extract powder.
Table 1: Grouping of the animals
S. No Groups Dose Sex Number Treatment schedule
1 Normal (G1) Portable H2O 10ml/kg/body
weight Female 6 Portable H2O treatment 10ml/kg/bw, for 7 days
2 Control (G2) GSM- RF
radiation 900mHz induction for
5-6hrs Female 6 Induction of radiation, 900mHz for 5-6hrs for 7+7 days followed by vehicle
3 Pre- treatment low dose
(G3) 100mg/kg/bw, DB Female 6 Treatment of drug+ induction of RF radiation for 7 days
4 Pre- treatment high dose
(G4) 200mg/kg/bw, DB Female 6 Drug treatment of high dose
+ induction of RF radiation for 7 days
5 Post- treatment low dose
(G5) 100mg/kg/bw, DB Female 6 Induction of RF radiation+ treatment of DB for 7 days
6 Post- treatment high dose
(G6) 200mg/kg/bw, DB Female 6 Induction of radiation+ treatment of DB for 7 days

The animals were started with treatment protocol simultaneously, with the pre-treatment dosing without any induction, on the 11th day the animals were subjected to the induction of RFR modulation. Simultaneously the control animals were exposed to the induction. And rest 2 groups were treated with post treatment. At the end of the study on 21st day the animals were sacrificed using diethyl ether in anesthesia chamber. The vital organs such as liver, uterus, ovaries, heart & kidneys were collected for the protein estimation followed by histopathology. And the bone marrow cells were flushed out of both the femurs with PBS & FBS for the single cell gel-electrophoresis (comet assay) and micronucleus assay.

MN Assay:
The bone marrow was collected from both the femurs using PBS and FBS respectively. These samples were centrifuged at 4000 rpm for 10min. The formed pellets were collected using Eppendorf tips and the smears were made on the microscopic glass slides and were allowed to air dry for 5min and followed by fixation with methanol. The glass slides were stained using Giemsa stain and may-Grunwald's stain. The slides were allowed to dry and observed under the oil immersion objective to determine the formation of micronuclei and MN frequency
Scoring the number of polychromatic erythrocytes and normochromatic erythrocytes in 100 cells per animal was recorded and MNPCE frequency was calculated.
MN frequency = n/N x 100
Observations made on Micronucleus Assay were found that among the treated groups, all the groups have shown protective against GSM-RFR induced radiations, the group G4 has shown lowest % of MN frequency whereas, all other treated groups (G3,G5,G6) results lie on the same range.




Table 2:
Sl. No Groups Polychromatic erythrocytes/total Polychromatic erythrocyte/ normochromatic erythrocyte % MN frequency
1 G1 0.83 ±0.02 8.42 ± 1.43 0.08 ± 0.07
2 G2 0.73 ± 0.02 ** 3.48 ± 0.41 ** 5.63± 0.34 **
3 G3 0.80 ± 0.01## 5.30 ± 1.79## 3.04 ± 0.14##
4 G4 0.82 ± 0.03##* 5.08 ± 1.01##* 1.38±0.41##*
5 G5 0.86 ± 0.03## 6.39 ± 1.79## 3.34 ± 0.14##
6 G6 0.89 ± 0.01##* 7.78 ± 0.78 ##* 3.76 ± 0.23##*
Values given as Mean ±SD, n=6, **p<0.01 normal vs ##p<0.01vs treated groups
The results of the genotoxic studies for GSM- RFR 900MHz was induced for G2 vs. other groups are as follows. All the data were interpreted in the form of mean ±SD with one-way ANOVA and multiple comparison a form of Tukeys-test. The significant values were expressed by **p<0.01 and ##p<0.05 respectively.
The micronuclei formation was assessed and % MN frequency was calculated the animal groups were treated with different high-low concentrations of Desmostachya bipinnata extract. The formation of micronuclei was significantly decreased when compared to the treated groups (p<0.01). Whereas in the case of irradiated post-treated groups there was a slight increase in the micronuclei formation. The present study on the Desmostachya Bipinnata an aqueous extract was believed to show its DNA protective activity.
The results of MN assay illustrates that there is a significant increase of the % MN frequency and formation of the micronuclei in each group concerning the normal. The group (G2) has shown significant increase in % MN frequency when compared to the normal G1 group. Whereas in the treated groups such as G3, G4, G5, & G6 there were no significant increase in the % mitotic frequency. Among the treated groups, all the groups have shown protective against GSM-RFR induced radiations, the group G4 has shown lowest % of MN frequency whereas, all other treated groups (G3,G5,G6) results lie on the same range. In-vivo, may be due to the presence of phytochemicals constituents such as polyphenolic compounds, flavonoids, phenolic acids which have been shown to neutralize various free radicals such as ROS, OH. ROO, H2O2 by various pathways like metal chelation and electron donation as a reducing agent.

Preparation of slides for single cell gel-electrophoresis (comet assay):
Microscopic slides were chilled before coating them. Later the slides were washed with 70% ethanol and they were coated with 0.6% of NMA by 1ml of a pipette and the slides were covered with a rectangular cover slip and chilled for 5-10min so that a thin and uniform coat is formed. The cover slips were removed carefully so that it does not damage the coating. This was served as the primary coating The second coating was done by taking 0.7g of LMA in 70 ml of distilled water the solution was microwaved for 2min until the clear solution was obtained. Further, the second coating was done by taking the 150μL of LMA in a pipette and 100μL of cell suspension, was coated upon the primary coat and then the slides were covered with microscopic cover slips and were chilled for about 5-10 min until the uniform coat was obtained.
Further, the slides were incubated in lysis buffer overnight in a big Petri plate wrapped with aluminum foil in a dark palate. The slides were submerged in an electrophoresis unit horizontally containing an alkaline electrophoresis buffer. The electrophoresis was run for 20min, 16V, and 300mA. Slides were neutralized for 2min in neutralizing buffer. The slides can be stored for up to a week if slides are not analyzed immediately.
Before the fluorescent imaging, the slides were stained with ethidium bromide stain for 5min 3 drops of stain per slide. Observe the staining in the UV transilluminator for the presence of excess stain, the excess stain was de-stained with distilled water. Slides were observed under 40x objective of a fluorescent microscope (Olympus) by using a CCD camera. Images of 50 randomly selected cells were captured and the images were analyzed by Open COMET IV software. The software provides an automatic examination of the comets.
The tail intensity and % of head DNA were noted.
Tail moment= tail length* %tail DNA÷100
Olive movement = (tail mean-head mean) * %DNA in the tail
Micronuclei, along with other nuclear anomalies, such as nucleoplasmic bridges and nuclear buds, are considered useful biomarkers that indicate genetic damage and chromosomal instability.
The % olive & tail moment of the GSM-RFR exposed animals were significantly increased (**p<0.01) with the G2 group when compared with the G1 and decreased when treated with Desmostachya bipinnata extract. The treatment group G4 has shown to have protective action against GSM-RFR mediated modulations; however, the other treatment groups such as G3, G5, G6 remains in a similar range.
Table 3: Table of olive & tail movement and tail intensity of different groups
Sl. No Groups Olive movement Tail length Tail intensity
1 G1 1.12 ± 0.02 0.15 ± 1.4 0.19 ± 0.04
2 G2 98.37 ± 13.14** 110.16 ± 10.09** 95.39 ± 0.05
3 G3 6.08 ± 0.04## 13.7 ± 3.08## 6.65 ± 1.25##
4 G4 5.76 ± 11.48##* 12.80 ± 0.02##* 4.65 ± 1.15##*
5 G5 9. 08 ± 3.38## 11.10 ± 5.59## 7.38 ± 4.48##
6 G6 10.08 ± 1.13##* 10.18 ± 2.28 ##* 5.58 ± 2.25##*
Values are given as Mean ± SD, n=6, **p<0.01, vs normal group, ##p<0.05 vs treated groups
Data of the comet assay shows that there was a significant increase in the % olive and tail moment in bone marrow cells of irradiated mice (control) G2 group compared to the normal group G1 & treatment groups G3, G4, G5, G6.
Control (G2) group, has shown to have a significant increase in the % olive and tail moment when compared to the normal (G1). Similarly, treatment groups such as G3, G4, G5, G6 has shown decrease in the olive & tail moment of the GSM-RFR exposed animals. The % of olive and tail moment in the treated groups were exposed to RFR modulations G3, G4, G5, & G6 when compared to the normal (G1) has shown to have slight increase in the olive moment.
Fig. 1 illustrates images (Images given as A, - normal, BC - control, D -PTLD, E - PTHD, F -PSLD, G -PSHD). The figure represents pictures showing the DNA damage and formation of the olive and tail movement of the bone marrow cells of the RFR induction. Images explaining both pre and post treated groups with 100mg/kg and 200mg/kg respectively.
Fig. 2 illustrates data represented as % olive movement vs groups. The olive movement was significantly increased in the G2 group when compared to the G1 and decreased with other Desmostachya bipinnata extract treated groups.
Data represented as % olive movement vs groups. The olive movement was significantly increased in the G2 group when compared to the G1 and decreased with other Desmostachya bipinnata extract treated groups. Data was expressed in the form of mean ±SD. Data was replicated as mean ±SD. Data was analysed by one-way ANOVA followed by multiple comparison vs control group, the method of Tukey. (**p<0.01) control group vs ( ##p<0.05)
Fig. 3 illustrates data represented as % tail movement/ tail length vs groups. The tail movement/ tail length was significantly increased in the G2 when compared to the G1 and decreased with other Desmostachya bipinnata extract treated groups. Data represented as % tail movement/ tail length vs groups. The tail movement/ tail length was significantly increased in the G2 when compared to the G1 and decreased with other DESMOSTACHYA BIPINNATA EXTRACT treated groups. Data was replicated as mean ±SD , data was analysed by one-way ANOVA and multiple comparison vs control group a method of Tukey (**p<0.01) vs normal and (**p<0.05) vs treated groups.
Fig. 4 illustrates the effect of the Desmostachya Bipinnata Extract treated groups against the RFR exposed group. The G2 group was shown to have a significant increase in the %MN frequency when compared to the G1 and other treated groups. Data was replicated as mean ±SD. Data was analysed by one-way ANOVA and also by multiple comparison vs control and group a Tukey's method where (**p<0.01) vs (##p<0.01) normal.
The radio frequency radiation induced free radical synthesis results in the progression of the reactive oxygen species (ROS) and lipid peroxidation triggers cell damage and apoptosis. The ROS formed by the action of the oxidative imbalance promotes DNA damage and changes in the gene expression.
Decades of research on the RFR- EMR have led to several outcomes regarding the possible adverse effects of cognitive impairment, infertility, genetic damage and pre-maturation of the cells. The need for herbal medicines is relatively high than ever before due to their purity, potency, lack of side effects, and society's optimism in the herbal medicines and their attributes.
The % olive & tail moment of the GSM-RFR exposed animals were significantly increased (**p<0.01) with the G2 group when compared with the G1 and decreased when treated with Desmostachya bipinnata extract. The treatment group G4 has shown to have protective action against GSM-RFR mediated modulations; however, the other treatment groups such as G3, G5, G6 remains in a similar range.
Based on the ethnopharmacological data, we choose Desmostachya Bipinnata to continue our efforts to validate the efficacy and therapeutic safety of the traditional herb. Besides that, these herb sections were used to treat the pregnant uterus as a galactagogue, astringent, aphrodisiac, anti-inflammatory, anti-asthmatic, antipyretic, analgesic, diuretic, and sedative. The tissue damage occurs due to various exogenous factors by the lipid peroxidation and isoprostanes which in turn increases in the composition & functionality of aldehydes or protein carbonyls generated by the protein oxidation and oxidised base adducts formed by DNA oxidation.
Desmostachya bipinnata extract rendered promising free radical scavenging and antioxidant activity on pU19 DNA and yeast cells respectively as well as considerable defense activity against HU- induced DNA damage. Thus, the selected compound was shown DNA protective activity in- vitro.
The main purpose of this present study was to evaluate the potential anticlastogenicity of the Desmostachya Bipinnata against radiation-induced DNA damage. We assessed the genotoxicity mainly by the comet assay through dose-dependent DNA damage and control exposed animals were effectively found with the genetic damage. The drug was given in a graded dose of 100mg/kg/bw as a low dose and 200mg/kg/bw high dose respectively with both pre and post-radiation-induced treatment according to the acute oral toxicity studies. The control group animals exposed with RFR modulation for 5-6 hrs daily were shown to have a toxic effect and significant, p<0.01 and p<0.05.
In the 2nd parameter, the micronuclei formation was assessed and % MN frequency was calculated the animal groups were treated with different high-low concentrations of Desmostachya bipinnata extract. The formation of micronuclei was significantly decreased when compared to the treated groups (p<0.01), whereas in the case of irradiated post-treated groups there was a slight increase in the micronuclei formation. The present study on the Desmostachya Bipinnata an aqueous extract was believed to show its DNA protective activity In-vivo, may be due to the presence of phytochemicals constituents such as polyphenolic compounds, flavonoids, phenolic acids which have been shown to neutralize various free radicals such as ROS, OH. ROO, H2O2 by various pathways like metal chelation and electron donation as a reducing agent.

Summarizing the present invention, a pharmaceutical composition for inhibiting damage to chromosomes comprising Desmostachya Bipinnata extract - 0.5-80%; diluents from 0% to 78.0%; lubricants from 0% to 3.0%; binders from 0% to 5.0%; and other pharmaceutically acceptable excipients. The composition is preferably in the form of a tablet or capsule or parenteral. The active ingredient used in the present pharmaceutical composition is Desmostachya Bipinnata extract powder. The solid dosage form tablet or capsule is prepared using active ingredient and pharmaceutically acceptable excipients selected from the group comprising of diluents, lubricants, glidants and other pharmaceutically acceptable excipients or adjuvants.
The diluent is selected from the group comprising of mannitol, microcrystalline cellulose, lactose, starch, dibasic calcium phosphate anhydrous, tribasic calcium phosphate, kaolin, sucrose, precipitated calcium carbonate, sorbitol, maltodextrin, powdered cellulose, micro crystalline cellulose and other materials known for such property. The lubricant is selected from the group comprising of stearic acid, sodium stearyl fumarate, polyethylene glycol, magnesium stearate, calcium stearate, talc, zinc stearate, hydrogenated castor oil, silica, colloidal silica, corn-starch, calcium silicate, magnesium silicate, silicon hydrogel and other materials known for such property.
The binder is selected from the group comprising of polyvinylpyrrolidone, hydroxypropyl methylcellulose, acacia, alginic acid, hydroxy propyl cellulose, carboxymethylcellulose sodium, compressible sugar, ethylcellulose, gelatin, liquid glucose, methylcellulose, pregelatinized starch and other materials known to one of ordinary skill in the art. The pharmaceutical composition may optionally be coated with functional and/or non-functional layers comprising film-forming polymers includes ethylcellulose, hydroxypropyl methylcellulose, hydroxypropylcellulose, polyvinyl alcohol, polyvinyl acetate methylcellulose, carboxymethyl cellulose, hydroxymethylcellulose, hydroxyethylcellulose, cellulose acetate, hydroxypropyl methylcellulose phthalate, cellulose acetate phthalate, cellulose acetate trimellitate; waxes; methacrylic acid polymers and the like.
The pharmaceutical composition may optionally comprising alkalising agent(s) in the form of a 1% aqueous solution or suspension alkalising agent may be selected from the group comprising sodium dihydrogen phosphate disodium dihydrogen phosphate, trisodium phosphate, sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, potassium hydrogen phosphate, dipotassium hydrogen phosphate, tripotassium phosphate and other materials known for such property.
A method for preparing the Desmostachya Bipinnata extract powder comprising drying the whole plant Desmostachya Bipinnata in shade for about 10 days; pulverizing into coarse powder; extracting the active material by water extraction process and kept in a shaking incubator for 2- 3hrs at 200rpm, filtering the extract and evaporated to dry powder.
Our results are as par with the previously reported an radioprotective activity by various authors such as, (Ayumi Yamamato et.al.,) reported the radioprotective activity of blackcurrant extract evaluated by in vitro micronucleus and gene mutation assays in TK6 human lymphoblatoid cells, he concluded the study with the evidences of antigenotoxic and radioprotective action of black current extract (BCE) was due to the presence of the high concentrations polyphenolic compounds along with anthocyanin, and L-ascorbic acid which exerts genoprotective activity with free radical scavenging.





, Claims:We claim:
1. A pharmaceutical composition for inhibiting damage to chromosomes comprising
Desmostachya Bipinnata extract - 0.5-80%;
diluents from 0% to 78.0%;
lubricants from 0% to 3.0%;
binders from 0% to 5.0%; and
other pharmaceutically acceptable excipients

2. The pharmaceutical composition according to claim 1, wherein the composition is preferably in the form of a tablet or capsule or parenteral.

3. The pharmaceutical composition according to claim 1, wherein the active ingredient used in the present pharmaceutical composition is Desmostachya Bipinnata extract powder.

4. The pharmaceutical composition according to claim 1, wherein the solid dosage form tablet or capsule is prepared using active ingredient and pharmaceutically acceptable excipients selected from the group comprising of diluents, lubricants, glidants and other pharmaceutically acceptable excipients or adjuvants.

5. The pharmaceutical composition according to claim 1, wherein diluent is selected from the group comprising of mannitol, microcrystalline cellulose, lactose, starch, dibasic calcium phosphate anhydrous, tribasic calcium phosphate, kaolin, sucrose, precipitated calcium carbonate, sorbitol, maltodextrin, powdered cellulose, micro crystalline cellulose and other materials known for such property.



6. The pharmaceutical composition according to claim 1, wherein lubricant is selected from the group comprising of stearic acid, sodium stearyl fumarate, polyethylene glycol, magnesium stearate, calcium stearate, talc, zinc stearate, hydrogenated castor oil, silica, colloidal silica, corn-starch, calcium silicate, magnesium silicate, silicon hydrogel and other materials known for such property.

7. The pharmaceutical composition according to claim 1, wherein binder is selected from the group comprising of polyvinylpyrrolidone, hydroxypropyl methylcellulose, acacia, alginic acid, hydroxy propyl cellulose, carboxymethylcellulose sodium, compressible sugar, ethylcellulose, gelatin, liquid glucose, methylcellulose, pregelatinized starch and other materials known to one of ordinary skill in the art.

8. The pharmaceutical composition according to claim 1, wherein may optionally be coated with functional and/or non-functional layers comprising film-forming polymers includes ethylcellulose, hydroxypropyl methylcellulose, hydroxypropylcellulose, polyvinyl alcohol, polyvinyl acetate methylcellulose, carboxymethyl cellulose, hydroxymethylcellulose, hydroxyethylcellulose, cellulose acetate, hydroxypropyl methylcellulose phthalate, cellulose acetate phthalate, cellulose acetate trimellitate; waxes; methacrylic acid polymers and the like.

9. The pharmaceutical composition according to claim 1, wherein may optionally comprising alkalising agent(s) in the form of a 1% aqueous solution or suspension alkalising agent may be selected from the group comprising sodium dihydrogen phosphate disodium dihydrogen phosphate, trisodium phosphate, sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, potassium hydrogen phosphate, dipotassium hydrogen phosphate, tripotassium phosphate and other materials known for such property.




10. A method for preparing the Desmostachya Bipinnata extract powder comprising;
(a) drying the whole plant Desmostachya Bipinnata in shade for about 10 days;
(b) pulverizing into coarse powder;
(c) extracting the active material by water extraction process and kept in a shaking incubator for 2- 3hrs at 200rpm.
(d) filtering the extract and evaporated to dry powder.

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