Oxidative DNA damage preventive activity and antioxidant potential of plants used in Unani system of medicine
© Kalim et al; licensee BioMed Central Ltd. 2010
Received: 28 July 2010
Accepted: 16 December 2010
Published: 16 December 2010
There is increasing recognition that many of today's diseases are due to the "oxidative stress" that results from an imbalance between the formation and neutralization of reactive molecules such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can be removed with antioxidants. The main objective of the present study was to evaluate the antioxidant activity of plants routinely used in the Unani system of medicine. Several plants were screened for radical scavenging activity, and the ten that showed promising results were selected for further evaluation.
Methanol (50%) extracts were prepared from ten Unani plants, namely Cleome icosandra, Rosa damascena, Cyperus scariosus, Gardenia gummifera, Abies pindrow, Valeriana wallichii, Holarrhena antidysenterica, Anacyclus pyrethrum, Asphodelus tenuifolius and Cyperus scariosus, and were used to determine their total phenolic, flavonoid and ascorbic acid contents, in vitro scavenging of DPPH·, ABTS·+, NO, ·OH, O2.- and ONOO-, and capacity to prevent oxidative DNA damage. Cytotoxic activity was also determined against the U937 cell line.
IC50 values for scavenging DPPH·, ABTS·+, NO, ·OH, O2.- and ONOO- were in the ranges 0.007 ± 0.0001 - 2.006 ± 0.002 mg/ml, 2.54 ± 0.04 - 156.94 ± 5.28 μg/ml, 152.23 ± 3.51 - 286.59 ± 3.89 μg/ml, 18.23 ± 0.03 - 50.13 ± 0.04 μg/ml, 28.85 ± 0.23 - 537.87 ± 93 μg/ml and 0.532 ± 0.015 - 3.39 ± 0.032 mg/ml, respectively. The total phenolic, flavonoid and ascorbic acid contents were in the ranges 62.89 ± 0.43 - 166.13 ± 0.56 mg gallic acid equivalent (GAE)/g extract, 38.89 ± 0.52 - 172.23 ± 0.08 mg quercetin equivalent (QEE)/g extract and 0.14 ± 0.09 - 0.98 ± 0.21 mg AA/g extract. The activities of the different plant extracts against oxidative DNA damage were in the range 0.13-1.60 μg/ml. Of the ten selected plant extracts studied here, seven - C. icosandra, R. damascena, C. scariosus, G. gummifera, A. pindrow, V. wallichii and H. antidysenterica - showed moderate antioxidant activity. Finally, potentially significant oxidative DNA damage preventive activity and antioxidant activity were noted in three plant extracts: C. icosandra, R. damascena and C. scariosus. These three plant extracts showed no cytotoxic activity against U937 cells.
The 50% methanolic extracts obtained from different plant parts contained significant amounts of polyphenols with superior antioxidant activity as evidenced by the scavenging of DPPH·, ABTS·+, NO, ·OH, O2.- and ONOO-. C. icosandra, R. damascena and C. scariosus showed significant potential for preventing oxidative DNA damage and radical scavenging activity, and the G. gummifera, A. pindrow, V. wallichii, H. antidysenterica, A. pyrethrum, A. tenuifolius and O. mascula extracts showed moderate activity. The extracts of C. icosandra, R. damascena and C. scariosus showed no cytotoxicity against U937 cells. In conclusion, these routinely used Unani plants, especially C. icosandra, R. damascena and C. scariosus, which are reported to have significant activity against several human ailments, could be exploited as potential sources of natural antioxidants for plant-based pharmaceutical industries.
The World Health Organization estimates that 80% of the world's inhabitants rely mainly on traditional medicines for their health care . Herbs contain some of the most powerful natural antioxidants and are highly prized for their antioxidant and anti-ageing effects.
Natural products offer an untold diversity of chemical structures. These compounds often serve as lead molecules, the activities of which can be enhanced by chemical manipulation and by de novo synthesis [2, 3]. To date, many medicinal plants have proved successful in combating various ailments, leading to mass screening for their therapeutic components.
Antioxidants are widely used as ingredients in dietary supplements and are exploited to maintain health and prevent oxidative stress-mediated diseases such as cancer, atherosclerosis, diabetes, inflammation and ageing. Recently, many antioxidants have been isolated from different plant materials [4–6]. Natural antioxidants are also in high demand for application as nutraceuticals and as food additives because of consumer preferences [7, 8]. In addition to their uses in medicine, these compounds are used in industry e.g. as preservatives in food and cosmetics and for preventing the degradation of rubber and gasoline. Antioxidants are also used as additives to help guard against food deterioration. Among natural antioxidants, plant polyphenols+ are especially important . Today, the search for natural compounds rich in antioxidant, anticancer and antimicrobial properties is escalating because of their importance in controlling many chronic disorders such as cancer and cardiovascular diseases . It has been estimated that approximately two-thirds of anticancer drugs approved worldwide up to 1994 were derived from plant sources .
It is increasingly being realized that many of today's diseases are due to the "oxidative stress" that results from an imbalance between the formation and neutralization of prooxidants . These excess free radicals react with biological macromolecules such as proteins, lipids and DNA in healthy human cells and this results in the induction of carcinogenesis, atherosclerosis, cardiovascular diseases, ageing and inflammatory diseases [11, 12]. These harmful radicals have to be eliminated from biological systems by enzymes such as superoxide dismutase, catalase and peroxidase, or compounds such as ascorbic acid, tocopherol and glutathione, which possess antioxidant properties.
Unani medicine, a form of traditional medicine widely practiced in India and the rest of the Indian subcontinent, is orientated towards prevention, health maintenance and treatment. Herbal products are regularly used in traditional medicines such as Ayurveda and Unani, which strengthen body defences . Unani therapies cure the diseases without such side effects even after they have been consumed for a long time with a wide spectrum of therapeutic activity. Unani therapies are known to be relatively economic and are most popular amongst people because they are safe and have time-tested efficacy. They contain vitamins, minerals, active steroids, alkaloids, glycosides and tannins as well as a variety of antioxidants in a biologically natural state.
Properties of Unani plants used in this study
Class of compound
Name of compound
Seed3, leaves, flower
It is vata and kapha suppressant, a good pain reliever, also a good antibacterial and antiwormal, reduces pus formation in the wounds, helpful in convulsions, has a good effect on digestive tract and improves indigestion condition in the body, increases sweating in the body
Cleomiscosin A, C,
Gulkhand made by the mixture of rose petals and white sugar in equal proportion act as the tonic and laxative, used as herbal tea in the treatment of cold and cough
Components of Essential oil
Flavonol and their glycosides
Citronellol, Geraniol, Linalool etc.
Glycosides of Kaempferol and Quercetin
Intestinal disorders, astringent, diaphoretic, diuretic, desiccant, cordial, and stomachic properties, treatment of gonorrhea
Some volatile compounds reported till date from the oil are atchoulanol, selina-4, etc.
Kapha skin disease, indigestion, worm infestation, diarrhea and infections, the resin has antiseptic property
Bark3, leaves, trunk
Disorders with inflammatory system
Potential rich sources up to 5% of bark weight
Antisplasmodic, stimulant, calmative and stomachic, useful in diseases of eye and liver, used as a remedy for hysteria, hypochondriasis, nervous unrest and emotional arrest, also useful in clearing voice and acts as a stimulant in advance stage of fever and nervous disorder
Essential oil and volatile oil
0.3-1% volatile oil content
Valtrate, didrovaltrate, acetovaltate, etc.
The bark is used as an astringent, anthelmintic, antidontalgic, stomachic, febrifuge, antidropsical, diuretic, in piles, colic, dyspepsia, chest affections and as a remedy in diseases of the skin and spleen, use as a well-known drug for amoebic dysentery and other gastric disorders
Stimulant, sialogogue, and rubefacient properties
Tonic, aphrodisiac, yield a lot of mucilage with water and form a jelly that is supposed to be nutritious and useful in diarrhea, dysentery, and chronic fever
Bitter principle and a volatile oil
Onion weed/Jangli pyaz
As diuretic and on inflammation
Luteolin and its glycosides
Plant materials and extraction procedure
Plants were collected from, and authenticated by, a Unani medical practitioner in Kolkata, India who regularly prescribes these materials. The different plant parts were shade-dried at room temperature (25°C) with occasional turning of the plants upside down for 5-7 days, and then ground to coarse powder with a mechanical grinder. The powdered plant materials (2 g) were extracted with 50 ml of aqueous methanol (50:50) for three consecutive days with intermittent stirring (1 h stirring at every 12 h interval) using magnetic stirrer until the extracts were light colored. The combined extracts were filtered and evaporated under reduced pressure in a rotary vacuum evaporator (Eyela NVC-2100--Rotary Evaporator, water bath temperature maintained at 40°C and 356 mm Hg, Eyela NCB-1200 Chiller unit temperature maintained at 7.5°C). The aqueous layer was lyophilized (at -45°C) and the dry powder was stored at -20°C for future use.
2, 2-Diphenyl-1-picrylhydrazyl (DPPH), thiobarbituric acid (TBA), Folin--Ciocalteu's phenol reagent, butylated hydroxytoluene, agarose and ethidium bromide were purchased from Sigma-Aldrich, USA. 2,2'-Azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), potassium persulphate, aluminium chloride, iron (III) chloride, and iron (II) sulphate were obtained from MP Biomedicals, USA. 2-Deoxy-D-ribose and ascorbic acid were procured from Himedia Laboratories Pvt. Ltd., Mumbai, India. The QIAprep Spin Miniprep Kit was purchased from Qiagen, Germany. All other chemicals and reagents used were of analytical grade.
For all the analytical studies, absorbance was measured using a Shimadzu UV-Visible Pharmaspec 1700 spectrophotometer.
Determination of total phenolic content (TPC)
The total phenolic contents of the 50% methanolic plant extracts were determined with gallic acid as a positive standard . Aliquots of test samples (100 μl) were mixed with 2 ml 2% Na2CO3 and incubated at 25°C for 2 min. After incubation, 1:1 (v/v) Folin-Ciocalteu's phenol reagent was added and the contents were mixed vigorously. The mixture was allowed to stand at 25°C for 30 min and the absorbance was measured at 720 nm. The same procedure was repeated with all standard gallic acid solutions and a standard curve was obtained. The total polyphenolic contents of the extracts were expressed in terms of gallic acid equivalents (GAE) of the plant sample.
Determination of total flavonoid content (TFC)
The total flavonoid content was determined using quercetin as a positive standard and expressed in terms of quercetin equivalents (QEE) in mg/g plant sample . NaNO2 (150 μl, 5% w/v) was added to tubes containing plant extracts in 2.5 ml distilled water. The contents were mixed thoroughly and allowed to stand for 5 min at ambient temperature, then 1.5 ml of 10% (w/v) AlCl3 were added and the mixture was allowed to stand for another 6 min. The solution was immediately mixed after addition of 1 ml 1 M NaOH. After 10 min, the absorbance was measured at 510 nm.
Determination of total ascorbic acid (ASC)
ASC of plant extracts were determined according to Roe and Kuether  after brief modification. Blanks, standards and samples were prepared in triplicate to measure ASC. Ascorbic acid (AA) standards (0-10 mM) or samples were precipitated with 10% trichloroacetic acid followed by centrifugation. In 500 μL of supernatant, 100 μL of DTC reagent (2,4-dinitrophenylhydrazine 3%, thiourea 0.4%, and copper sulfate 0.05%) prepared in 9N sulfuric acid, was mixed and incubated at 37°C for 3 h. After the addition of 750 μL of 65% (v/v) sulfuric acid, the absorbance was recorded at 520 nm. A standard curve was prepared with AA standards, and ASC was expressed as mg AA/g of plant sample.
Determination of free radical scavenging activity
The radical scavenging activities of the plant extracts in the range 0-200 μg/ml were evaluated using DPPH·. Stock solutions of plant extracts were prepared at a concentration of 10 mg/ml and a freshly-prepared DPPH solution (100 mM) was used as described previously .
Determination of hydroxyl radical scavenging activity
The ·OH scavenging assay was performed as standardized before . The reaction mixture consisted of different concentrations (0-100 μg/ml) of plant extract, 3.6 mM deoxyribose, 0.1 mM EDTA, 0.1 mM L-ascorbic acid, 1 mM H2O2 and 0.1 mM FeCl3.6H2O, and the volume was made up to 500 μl with 25 mM phosphate buffer, pH 7.4. This mixture was incubated for 1 h at 37°C, 500 μl of 1% TBA and 500 μl of 1% TCA were added, and the mixture was heated in a boiling water-bath for 15 min and then cooled. The absorbance was measured at 532 nm. The control reaction contained no test sample, and quercetin (20 μg/ml) was used as a standard. Percentage RSC was calculated as described above.
Determination of peroxynitrite scavenging activity
Peroxynitrite was synthesized by the method of Beckman et al. 1994 . Briefly, an acidic solution of 0.7 M H2O2 was mixed with an equal volume of 0.6 M potassium nitrite in an ice bath and an equal volume of ice cold 1.2 M NaOH was added. Granular MnO2 prewashed with 1.2 M NaOH was used to remove excess H2O2 and the reaction mixture was left at -20°C. The concentration of peroxynitrite generated was measured spectrophotometrically at 302 nm (ε = 1670 M-1 cm-1).
Peroxynitrite scavenging activity was measured according to Hazra et al. 2010 . The reaction mixture consisted of 0.1 mM DTPA, 90 mM NaCl, 5 mM KCl, 12.5 μM Evans Blue, plant extracts at various doses ranging from 0-300 μg/ml, and 1 mM peroxynitrite adjusted to a final volume of 1 ml with 50 mM phosphate buffer (pH 7.4). The reaction mixture was incubated at 25°C for 30 min and the absorbance was measured at 611 nm. The percentage peroxynitrite scavenging activity was calculated by comparing the results of the test and blank samples; gallic acid served as the reference compound. All tests were conducted six times. The IC50 values of the extracts were calculated by regression analysis.
Determination of non-enzymatic superoxide radical scavenging activity
Superoxide radical was generated in vitro by a non-enzymatic method involving the nicotinamide adenine dinucleotide-nitro blue tetrazolium-phenazine methosulphate (NADH-NBT-PMS) system following the procedure of Nishikimi et al. . NBT (150 μM in 0.02 M Tris buffer, pH 8.0) was added to 1 ml of NADH solution (50 μM of NADH in 0.02 M Tris buffer, pH 8.0) in the presence of various concentrations (0-50 μg/ml) of extracts. The reactions were initiated by adding PMS (15 μM) and the absorbance was at 560 nm was measured exactly 1 min later. Results were recorded as percentage inhibition. Quercetin at various concentrations was used as standard. All tests were performed six times.
Nitric oxide scavenging activity: concentration dependence
The scavenging activity against nitric oxide was assayed by the method of Marcocci et al. . Sodium nitroprusside (0.5 ml, 5 mM in 20 mM phosphate buffer, pH 7.4, previously bubbled with argon) was added to tubes containing 0.5 ml of different plant extracts of various concentrations (0-300 μg/ml) and incubated at 25°C for 150 min. At the end of the incubation, 1 ml of Griess reagent (equal volumes of 2% w/v sulphanilamide in 5% phosphoric acid and 0.2% w/v naphthylethylenediamine dihydrochloride) was added to each sample and the absorbance was measured at 546 nm against control samples (extracts incubated with only 20 mM phosphate buffer, pH 7.4) and referred to the absorbance of standard solutions of sodium nitrite treated in the same way with Griess reagent. Results were recorded as percentage nitrite formed. Quercetin at various concentrations was used as standard.
Prevention of oxidative DNA damage
This was determined as described previously . Plasmid DNA was isolated using a QIAprep Spin Miniprep Kit according to the manufacturer's instructions. Plasmid pBluescript II SK (-) (250 ng) was treated with FeSO4, H2O2 and phosphate buffer (pH 7.4) in final concentrations of 0.5 mM, 25 mM and 50 mM, respectively, and test extracts at different concentrations (0-2 μg/ml). The total reaction volume was set to 12 μl and the mixture was incubated at 37°C for 1 h. After the incubation, the extent of DNA damage and the preventive effect of the test samples were analyzed on 1% agarose gels at 70 V at room temperature. Quercetin (1 mM) was used as positive control.
Gels were scanned on a Gel documentation system (GelDoc-XR, Bio-Rad, Hercules, CA, USA). Bands were quantified using discovery series Quantity One 1-D analysis software (Bio-Rad).
In vitro cytotoxicity activity (MTT assay)
Where At = absorbance of test sample; Ab= absorbance of blank (medium); Ac= absorbance of control (cells).
All data were expressed as mean ± SD. Statistical analyses were performed using Microsoft Excel. The IC50 values were calculated by regression analysis. Values with p < 0.05 were considered statistically significant. The IC50 values were compared by paired t test (two-sided).
Total phenolic, flavonoid and ascorbic acid contents
Total phenolic, flavonoid and ascorbic acid contents of plant extracts
Total phenolic content
mg GAE/g plant extract1
Total flavonoid content
mg QEE/g plant extract1
Total ascorbic acid content
mg AA/g plant extract1
166.13 ± 0.56
172.23 ± 0.08
0.98 ± 0.218
142.23 ± 0.09
151.32 ± 0.51
0.82 ± 0.092
128.83 ± 0.32
118.93 ± 0.23
0.39 ± 0.017
82.72 ± 0.03
87.32 ± 0.13
0.49 ± 0.029
76.82 ± 0.13
63.82 ± 10.71
0.47 ± 0.079
72.13 ± 0.51
74.32 ± 0.21
0.55 ± 1.012
69.12 ± 0.35
60.42 ± 0.34
0.42 ± 0.077
62.89 ± 0.43
38.89 ± 0.52
0.37 ± 0.12
12.52 ± 0.57
12.11 ± 1.20
0.33 ± 0.073
15.74 ± 0.98
11.98 ± 0.74
0.14 ± 0.091
The mean values of total phenols ranged from 62.89 ± 0.43 to 166.13 ± 0.56 mg GAE/g, flavonoids from 38.89 ± 0.52 to 172.23 ± 0.08 mg QEE/g extract and ASC from 0.14 ± 0.091 to 0.98 ± 0.218 AA/g extract. The highest TPC was observed in C. icosandra (166.13 ± 0.56 mg GAE/g extract), followed by R. damascena (142.23 ± 0.09 mg GAE/g extract) and C. scariosus (128.83 ± 0.32 mg GAE/g extract). For TFC, C. icosandra (172.23 ± 0.08 mg QEE/g extract) showed the highest content, also followed by R. damascena (151.32 ± 0.51 mg QEE/g extract) and C. scariosus (118.93 ± 0.23 mg QEE/g extract). The ASC contents were 0.98 ± 0.21, 0.82 ± 0.092 and 0.39 ± 0.017 mg AA/g extract in C. icosandra, R. damascena and C. scariosus, respectively followed by other extracts.
DPPH· scavenging activity
IC50 values of plant extracts (μg/ml)
7.28 ± 0.37**
2.54 ± 0.04***
20.13 ± 0.01***
152.23 ± 3.51***
30.96 ± 0.98***
532.85 ± 15.93*
10.36 ± 0.02***
3.57 ± 0.11**
23.01 ± 0.03**
273.18 ± 3.52***
42.10 ± 0.82NS
637.57 ± 52.93**
11.10 ± 0.37**
6.27 ± 0.44**
18.23 ± 0.038***
240.31 ± 4.28***
28.85 ± 0.23***
590.23 ± 2.37**
82.33 ± 0.31***
11.62 ± 0.21**
34.33 ± 0.07***
45.39 ± 0.87***
890.32 ± 52.23***
84.23 ± 1.50**
19.10 ± 0.21***
31.43 ± 0.07***
286.59 ± 3.89***
74.54 ± 9.28***
987.42 ± 17.4***
86.61 ± 0.89**
21.26 ± 0.18***
37.92 ± 0.07***
78.35 ± 0.57***
943.12 ± 27.82***
98.84 ± 0.31***
29.92 ± 0.25***
29.23 ± 0.01**
211.34 ± 2.12***
83.49 ± 0.59***
880.51 ± 9.99***
467.10 ± 0.27***
31.76 ± 0.27***
41.22 ± 0.04***
83.49 ± 0.59***
1.137 ± 0.0031***
1.098 ± 0.0091***
47.82 ± 0.20**
537.87 ± 93.12**
3.114 ± 0.091***
2.006 ± 0.0021***
156.94 ± 5.28***
50.13 ± 0.04***
425.92 ± 78.12***
3.390 ± 0.0311***
3.21 ± 0.11
1.34 ± 0.08
7.42 ± 0.32
18.23 ± 0.42
41.98 ± 0.95
820.12 ± 27.34
ABTS·+ scavenging activity
The IC50 values of the plant extracts were also determined for ABTS·+ (Table 3). Significant activity was noted with C. icosandra, R. damascena and C. scariosus with 98.23% inhibition at 6.98 ± 0.07 μg/ml, 91.83% inhibition at 8.42 ± 0.13 μg/ml and 72% inhibition at 8.32 ± 0.09 μg/ml, respectively.
·OH scavenging activity
The ·OH scavenging potentials manifested by the different plant extracts were also evaluated by decreased formation of the chromogen in the Fenton reaction. The ·OH scavenging activities of the 50% methanolic extracts correlated with protection against DNA damage, as shown in Table 3. Best scavenging activity was noted with C. icosandra, R. damascena and C. scariosus which showed 67.08% inhibition at 34.54 ± 0.92 μg/ml, 69.7% inhibition at 23.48 ± 0.85 μg/ml and 67.2% inhibition at 29.33 ± 0.43 μg/ml, respectively.
Peroxynitrite scavenging activity
In all the extracts tested, peroxynitrite-scavenging activity was concentration dependent. The scavenging activities was, 72% inhibition at 766.08 ± 12.23 μg/ml, 78% inhibition at 993.72 ± 52.34 μg/ml and 69% inhibition at 814.2 ± 37.89 μg/ml for C. icosandra, R. damascena and C. scariosus respectively. Hence these three extracts have superior activity than that of gallic acid standard. Howerver, IC50 values of H. antidysenterica (880 ± 9.99 μg/ml) and G. gummifera (890 ± 52.23 μg/ml) were comparable to the standard (i.e. 820.12 ± 47.2 μg/ml) in peroxynitrite scavenging potential (Table 3).
Superoxide scavenging activity
As is evident from Table 3, the extracts of C. icosandra (73% inhibition at 45.20 ± 8.25 μg/ml), R. damascena (81% inhibition at 68.20 ± 7.23 μg/ml) and C. scariosus (78.23% inhibition at 45.23 ± 0.37 μg/ml) also caused considerable scavenging of superoxide anion in comparison to the reference compound quercetin. The IC50 values for the superoxide scavenging activities of extracts and the reference standard are shown in Table 3. As evident from results, C. scariosus (28.85 ± 0.23 μg/ml) was able to quench superoxide radicals more effectively than the reference compound quercetin (41.98 ± 0.95 μg/ml).
Nitric oxide scavenging activity
C. icosandra showed significant nitric oxide scavenging activity than that of other plant extracts having 69% inhibition at 210.07 ± 18.27 μg/ml. However modest scavenging activity was also noted with R. damascena (73.9% at 398.84 ± 52.1 μg/ml) and C. scariosus (72.24% at 350.85 ± 12.3 μg/ml) respectively. IC50 values were presented in Table 3 along with reference compound quercetin (at 18.23 ± 0.42 μg/ml).
Prevention of oxidative DNA damage by plant extracts
Correlation between the TPC or TFC with the antioxidant activity
Cytotoxic activity of three plants at two different concentrations
Doxorubicin 25 ng/ml
As a part of a concerted effort to develop herbal antioxidants from natural sources, we investigated several plants regularly prescribed in the Unani system of medicine against various human ailments. For initial free radical screening, DPPH assay followed by an ABTS assay was used which showed significant activity in C. icosandra, R. damascena and C. scariosus. To evaluate this potential more specifically, extracts were checked for ·OH scavenging and the highest activity was noted with C. icosandra, R. damascena and C. scariosus, corroborating the previous assay. Significant NO scavenging was noted with C. icosandra, followed sequentially by H. antidysenterica, C. scariosus and R. damascena. Peroxynitrite scavenging by C. icosandra, C. scariosus, and R. damascena was significantly greater than by the reference compound, whereas H. antidysenterica and G. gummifera showed similar activity to the standard compound. O2.- scavenging activity was also significant in the extracts of C. icosandra, C. scariosus and R. damascena. Taken together, these findings indicate that C. icosandra extract is a potential candidate for free radical scavenging followed by R. damascena and C. scariosus.
Phytochemical analysis revealed significant total phenolic and flavonoid contents in the extracts of these same three plants, C. icosandra, R. damascena and C. scariosus, and these correlated with their potential radical scavenging activities. Though the ASC of these three effective extracts were insignificant, however that of C. icosandra and R. damascena was little higher than of C. scariosus, indicating that the antioxidant potential of C. scariosus arises from its total phenolic and flavonoid contents. Other plant extracts are also reported to contain polyphenolic compounds and their antioxidant activities may be related to this .
Flavonoids are polyphenols naturally present in nearly all plant materials . Phenolic compounds are effective hydrogen donors, and this makes them good antioxidants . Flavonoids are a class of compounds that remain of great scientific and therapeutic interest, and their antioxidant activity has attracted most attention. Their high antioxidant potential is attributable to their capacity to scavenge harmful ROS and other free radicals that originate from various cellular activities and lead to oxidative stress . Plant-derived polyphenolic flavonoids are well known to exhibit antioxidant activity through a variety of mechanisms including scavenging of ROS, inhibiting lipid peroxidation and chelating metal ions . Hence their mechanism of action is multiple; it includes the inhibition of enzymes involved in ROS generation, chelating of trace metals such as free iron and copper, and the ability to reduce highly oxidizing free radicals by hydrogen donation, thus protecting us from serious diseases such as heart attacks, strokes and even cancers. In addition, ascorbic acid acting as a chain-breaking antioxidant impairs the formation of free radicals during the biosynthesis of intracellular and extracellular substances throughout the body, including collagen, bone matrix and tooth dentine .
Previous studies have reported that the seeds of C. icosandra contain coumarino-lignans such as cleomiscosin A, B, C and D, of which A and C are reported to be antioxidants [16, 35]. Collectively, these observations indicate that the free radical scavenging potential of C. icosandra seeds and protection they confer against oxidative DNA damage may be attributed to their phytochemical composition. Rose essential oil is widely used in perfumery and the cosmetic industry. In addition to its perfuming effects, it is reported to possess a wide range of biochemical activities. Petals of R. damascena contain flavonol aglycons like kaempferol, quercetin and its glycosides such as kaempferol glycosides, quercitrin etc., citronellol and geraniol as the major components of its essential oil as well as tocopherol and carotene [19, 36, 37]. Potential antioxidant activity of rose petals may be attributed for their diversified phytochemical contents, which are consistent with earlier reports [36–38]. C. scariosus roots contain compounds such as patchoulanol, isopatchoulenone, etc. as major components of its essential oil . Wei and Shibamoto  studied the antioxidant activities of major essential oils from several plants and reported that myristicin from parsley seeds, patchouli alcohol from patchouli, and citronellol from roses showed high antioxidant activities, which can be related to our study.
The Fenton reaction is a major physiological source of ·OH, which is produced near DNA molecules in the presence of transition metal ions such as iron and copper . As previous reports suggest, polyphenol-rich diets may decrease the risk of chronic diseases by reducing oxidative stress . The Fenton reaction is prevented by hydroxyl radical-scavenging flavonoids . Here, the capacities of all ten plant extracts to protect against oxidative DNA damage were checked against DNA strand scission by ·OH generated in Fenton reactions on pBluescript II SK (--) DNA. We conclude that a significant contributor to DNA damage prevention is the scavenging of ·OH by the extracts of C. scariosus, C. icosandra, R. damascena and H. antidysenterica at 0.13 μg/ml, 0.16 μg/ml, 0.2 μg/ml and 0.28 μg/ml, respectively; this was corroborated by densitometric analysis.
The three effective extracts, viz. C. icosandra, R. damascena and C. scariosus, were not cytotoxic in comparison to doxorubicin, and this appears consistent with their long history of use in the Unani system of medicine.
Unani plants that are reported to have significant activity against several human ailments showed superior antioxidant activity as evidenced by the scavenging of the free radicals DPPH·, ABTS·+, NO, ·OH, O2.- and ONOO-. Of the ten 50% methanolic plant extracts tested, three - namely C. icosandra, R. damascena and C. scariosus - showed potentially significantly capacity to prevent oxidative DNA damage and radical scavenging activity. The C. icosandra, R. damascena and C. scariosus extracts were not cytotoxic against U937 cells. To gain further insight into the basis of their antioxidant properties, TPC, TFC and ASC were determined. All three extracts showed significantly high TPC and TFC contents, which contribute to their antioxidant activities. In conclusion, these routinely used plants can be explored further as potential sources of natural antioxidants.
This work was supported by the network research grant from the Council of Scientific and Industrial Research (CSIR). MDK acknowledges the Department of Science and Technology (DST) for her fellowship. We are grateful to acknowledge BioMedES editorial services for copyediting the manuscript. We express our gratitude to Professor Siddhartha Roy, Director, IICB, for his help and support.
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