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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 10  |  Issue : 3  |  Page : 269-275

Effect of Vitex trifolia linn and Solanum nigrum Linn on oxidative stress and inflammation


Department of Pharm-Chemistry, KLE University's College of Pharmacy, Hubballi, Karnataka, India

Date of Web Publication5-Sep-2017

Correspondence Address:
A. H. M. Viswanathswamy
KLE University's College of Pharmacy, Vidyanagar, Hubballi - 580 032, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/kleuhsj.kleuhsj_11_17

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  Abstract 


Context: Phytochemical investigations of hydroalcoholic extracts of Vitex trifolia Linn and Solanum nigrum Linn suggest the presence of flavonoids, terpenoids, and steroids and have been traditionally claimed to be useful in the treatment of inflammation, fever, edema, and tuberculosis.
Aim: In the present study, hydroalcoholic extract of the plants was evaluated for its free radical scavenging capacity against hydroxyl, hydrogen peroxide, and nitric oxide radicals, and it's in vitro anti-inflammatory potential.
Materials and Methods: Hydroalcoholic extracts of V. trifolia L. and S. nigrum L. were studied at different concentrations for their scavenging ability against free radicals and their ability to protect against hypotonic solution-induced hemolysis on red blood cells and inhibition of protein denaturation. For evaluation, different in vitro assays were used.
Results: Results show that the plant extract possesses free radical scavenging capacity, membrane stabilizing capacity, and protein denaturation inhibition capacity which could be correlated to the traditional claim of the plant being used in the treatment of inflammation.
Conclusion: The study supports the traditional claim and the prior in vivo studies carried out. It also helps in understanding the possible mode of action for aforesaid activity.

Keywords: Anti-inflammatory, antioxidant capacity, in vitro, Solanum nigrum


How to cite this article:
Ankalikar AA, Viswanathswamy A. Effect of Vitex trifolia linn and Solanum nigrum Linn on oxidative stress and inflammation. Indian J Health Sci Biomed Res 2017;10:269-75

How to cite this URL:
Ankalikar AA, Viswanathswamy A. Effect of Vitex trifolia linn and Solanum nigrum Linn on oxidative stress and inflammation. Indian J Health Sci Biomed Res [serial online] 2017 [cited 2019 Nov 21];10:269-75. Available from: http://www.ijournalhs.org/text.asp?2017/10/3/269/213989




  Introduction Top


A normal protective response to any tissue injury is broadly termed as inflammation which involves a complex process of enzyme activation, release of mediators, and various fluids.[1] This causes activation of the phagocytes leading to production of O2 and nonfree radical species. This is a part of inflammatory response which causes damage by stimulating the matrix metalloproteinase. This aforesaid damage caused due to inflammation could be neutralized by scavengers and antioxidants.[2],[3],[4],[5]

The genus Vitex includes about 270 known species. Vitex trifolia is a shrub whose roots have been claimed to be having thermogenic expectorant, astringent, carminative, and anti-inflammatory activity. Reports are available of its use in leprosy, bronchitis, hair loss, and inflammations. The extracts of the plant are a source for diterepenes such as vitretifolin A, B, and C.[6],[7],[8],[9],[10]

Solanum nigrum is an herbaceous weed 30–45 cms in height. The extract of its fruits has been evaluated for antioxidant, cytotoxic, anti-diarrheal, chronic inflammatory conditions and antihyperlipidemic activity.[11],[12]

The present study was designed to investigate the possible mechanism of action and affirm the traditional claim of the plant being useful in the treatment of inflammation.


  Materials and Methods Top


Plant material and extract: The leaves of V. trifolia Linn were collected from the herbal garden of KLE University's College of Pharmacy, Vidyanagar, Hubballi, and the whole plant of S. nigrum Linn was collected around the District of Dharwad and Hubballi. The plants were authenticated from the Department of Botany, P. C. Jabin Science College, Hubballi. The plant material was collected, shade dried, and grinded in the mixer.

The powdered drug of the plant was stored in airtight polyethylene bag. For extraction, about 50 g of the powdered drug was weighed using an electronic balance and blended with about 200 ml 70% ethanol, refluxed for 1.5 h at 65°C–70°C. This cycle was repeated three times. Total percentage yield of the V. trifolia was found to be 22.94% and that of S. nigrum was found to be 9.045%.

Drugs and chemicals

Griess reagent and 2-deoxy-d-ribose were procured from Sigma-Aldrich. All other chemicals used were of analytical grade.

Spectroscopic readings

The absorbance reading was recorded on Shimadzu ultraviolet (UV)-1700 PharmaSpec UV–visible spectrophotometer.

The extracts were subjected to in vitro anti-inflammatory and antioxidant activity using different assay methods.

Hydrogen peroxide scavenging capacity

The ability of the extracts to scavenge hydrogen peroxide (H2O2) was determined according to Ruch et al.[13],[14]

The extracts and the standard ascorbic acid were tested at different concentrations, namely, 60, 80, 100, 120, and 140 μg/ml, which were prepared using distilled water and to this 0.6 ml of 40 mM H2O2 solution prepared in phosphate buffer pH 7.4 was added and kept undisturbed for incubation for a period of 10 min. All the solutions were kept for incubation for a period of 10 min, and absorbance of H2O2 solution at 230 nm was measured using a blank solution containing phosphate buffer without H2O2. All the readings were taken in triplicate. The percentage of H2O2 scavenging capacity was calculated using the formula:



(Where AC is the absorbance of the control and AS is the absorbance in presence of sample extracts or standard).

Nitric oxide radical activity

At the physiological pH 7.2, the extract produces nitric oxide radicals (NO). NO radical under aerobic condition interacts with oxygen present in the environment to generate nitrite ions measured using Griess reagent. NO radical was generated using 10 mM sodium nitroprusside at physiological pH. Different dilutions of the both the extracts as well as standard ascorbic acid (60, 80, 100, 120, and 140 μg/ml) were prepared in ethanol and incubated at 25°C for 10 min, after which 0.5 ml of Griess reagent was added to 0.5 ml of reaction mixture and absorbance was measured at 540 nm.[13],[15],[16]



(Where AC is the absorbance of the control and AS is the absorbance in presence of sample extracts or standard).

Hydroxyl radical scavenging activity

This method is based on “Fenton reaction” which involves in vitro generation of hydroxyl radicals using phenylhydrazine and measured using 2-deoxyribose. Solutions of both the extracts at different concentrations, namely, 60, 80, 100, 120, and 140 μg/ml were prepared in 50 mM phosphate buffer at pH 7.4, which contained 1 mM deoxyribose and 0.2 mM phenylhydrazine hydrochloride. This was followed by an incubation period of 1–4 h, after which 1 ml of 2.8% trichloroacetic acid and 1% w/v of thiobarbituric acid were added, and the reaction mixture was heated for 10 min in boiling water bath. On cooling, the absorbance was taken at 532 nm. The standard used in this method was ascorbic acid. The percentage scavenging capacity was calculated as follows:[17],[18]



(Where AC is the absorbance of the control and AS is the absorbance in presence of sample extracts or standard).

Phosphomolybdenum assay

Reduction of Mo (VI) to Mo (V) in acidic pH subsequently forms green phosphate. This assay tests the reduction capacity of the extract. To 0.3 mL of the extract 0.6M sulfuric acid, 28 mM sodium phosphate and 4 mm ammonium molybdate are added, and the tubes are incubated at 95°C for 90 min. Absorbance was measured at 695 nm against blank containing 0.3 ml of methanol. The antioxidant capacity is expressed as number of equivalent of ascorbic acid.[19]

Inhibition of albumin denaturation



Reaction mixture consisting of test extracts at different concentrations and 1% aqueous solution of bovine albumin fraction was prepared pH of the reaction mixture (6.3) and was adjusted using 1N HCl. Samples were incubated at 37 ° C for 20 min and then heated at 57 ° C for 20 min. The samples were then cooled and the turbidity was measured at 660 nm spectrophotometrically. Experiment was performed in triplicate for which similar volume of double-distilled water served as control. Percentage inhibition of albumin denaturation was calculated using the following formula:[20],[21],[22]

Heat-induced hemolysis

Part I: Fresh whole blood (10 ml) was collected and transferred into heparinized centrifuged tubes. The tubes were centrifuged at 3000 rpm for 10 min and washed three times with equal volume of normal saline. Volume of the blood was measured and reconstituted as 10% v/v suspension with normal saline.

Part II: The reaction mixture (2 ml) was prepared consisting of 1 ml of extract solution and 1 ml of 10% red blood cells suspension. For control instead of drug, saline was added. Standard drug aspirin was added to saline. The tubes were incubated in water bath at 56°C for 30 min. At the end, incubated tubes were cooled under running tap water, and the reaction mixture was centrifuged at 2500 rpm for 5 min. The supernatants were separated and absorbance taken at 560 nm. Readings were taken in triplicate.[23],[24],[25]

% membrane stabilization calculated using following formula.




  Results Top


Hydrogen peroxide scavenging capacity

The results of the in vivo assay show that with increase in concentration of the extract, the scavenging capacity of the extract also increases. At concentration of 60 μg/ml, the scavenging capacity of the hydroalcoholic extract of V. trifolia was seen to be 48.70%. As the concentration of the extract was increased to 80, 100, 120, and 140 μg/ml, scavenging capacity was seen to be 60.8%, 63.08%, 73.4%, and 74.8%, respectively, against H2O2 radical. On the other hand, standard ascorbic acid has exhibited 14.45%, 21.6%, 33.73%, 55.4%, and 59.03%, respectively, at 60, 80, 100, 120, and 140 μg/ml [Graph 1].



The hydroalcoholic extract of whole plant of S. nigrum shows a steady increase in scavenging capacity as the concentration of the extract increases. At the least concentration of 60 μg/mL, the hydroalcoholic extract of S. nigrum has shown 59.45% scavenging capacity with increase in concentration to 80, 100, 120, and 140 μg/ml; the scavenging capacity was seen to rise steadily to 67.71%, 70.06%, 73.4%, and 78.1%, respectively [Graph 2].



Nitric oxide scavenging capacity

The hydroalcoholic extract of leaves of V. trifolia is seen to be equally proficient as the standard ascorbic acid at lower concentration. While the extract shows 61.1% inhibition, ascorbic acid shows 58.2% inhibition at 60 μg/ml concentration. As the concentration of V. trifolia is increased to 80, 100, 120, and 140 μg/ml, the percentage inhibition increases to 67.9%, 78.6%, 83.4%, and 95.1%, respectively, which is higher as compared to the standard ascorbic acid at same concentration, namely, 62.9%, 70.8%, 75.7%, and 82.5%, respectively. Thus, it is seen that at higher concentration, the hydroalcoholic extract of V. trifolia is more proficient than the standard [Graph 3]. On evaluating the hydroalcoholic extract of S. nigrum for its % inhibition capacity at 60 μg/mL, the inhibition capacity was seen to be 73.84%, and with increase in concentration, the inhibition capacity was seen to be 73.8%, 75.7%, 78.6%, 81.5%, and 82.5%. Thus, as the concentration increases percentage, inhibition also increases, and at the concentration 140 μg/mL, both the extract and the standard ascorbic acid have shown similar inhibition capacity of 82.5% [Graph 4].



Deoxy-D-ribose scavenging assay/hydroxyl radical scavenging activity

At concentration of 60, 80, 100 and 120 μg/mL, the hydroxyl scavenging capacity of hydroalcoholic extract of leaves of V. trifolia was seen to be 21%, 39%, 49%, and 60%, respectively. The hydroalcoholic extract of S. nigrum at these concentrations has shown 15%, 37%, 55%, and 68% inhibition while standard ascorbic acid at same concentrations was seen to be 24%, 38%, 45%, and 59%. However, at high concentration of 140 μg/mL, the hydroxyl scavenging capacity of the hydroalcoholic extract of leaves of V. trifolia and the standard ascorbic acid was the same, namely, 71% and 72% inhibition capacity, respectively [Graph 5] while the scavenging capacity of hydroalcoholic extract of S. nigrum was slightly better, i.e., 76% [Graph 6].



Phosphomolybdenum assay

The total antioxidant capacity of extract of leaves of V. trifolia is same as that of ascorbic acid, i.e., equivalent to 132 mg/g ascorbic acid [Graph 7] while that of hydroalcoholic extract of S. nigrum has total antioxidant capacity equivalent to 68 mg/g of ascorbic acid at higher concentration [Graph 8].



The IC50 values for the hydroalcoholic extracts of S. nigrum, V. Trifolia, and the standard antioxidant ascorbic acid in different antioxidant assays were also calculated. The IC50 values for the hydroalcoholic extracts of V. trifolia for H2O2 scavenging capacity, NO scavenging capacity, and hydroxyl radical scavenging capacity were found to be 4.08, 0.259, and 3.22 ug/ml, respectively, while that for S. nigrum was found to be 1.59, 9.3, and 2.98 ug/ml, respectively. The IC50 values for the standard ascorbic acid were 0.793, 0.260, and 3.14 ug/ml, respectively [Table 1]. H2O2 scavenging capacity IC50 value of hydroalcoholic extract of V. trifolia is higher, i.e., 4.08 ug/ml as compared to the standard which is 0.793 μg/ml. The NO scavenging capacity of hydroalcoholic extract of S. nigrum is 9.3 μg/mL, which is higher than the capacity of standard which is 0.260 μg/ml. However, the hydroxyl radical scavenging capacity IC50 value for all three, namely, hydroalcoholic extracts of V. trifolia, S. Nigrum, and standard ascorbic acid is approximately the same, namely, 3.22, 2.98, and 3.14 ug/ml, respectively.
Table 1: Scavenging capacity of hydroalcoholic extract of Vitex trifolia and Solanum nigrum from various radicals

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Inhibition of albumin denaturation

The albumin denaturation capacity of the plant extracts was tested at 50, 100, 200, 400, 600, and 800 ug/ml concentrations, and it was seen that the extract of V. trifolia has shown inhibition of 17.01%, 24.44%, 42.68%, 53.12%, 64.01%, and 78.18% [Graph 9] while the extract of S. nigrum has inhibition capacity of 22.11%, 29.37%, 37.25%, 54.33%, 71.17%, and 89.97%, respectively [Graph 10]. The standard drug indomethacin has shown 98.98% inhibition indicating to be better at inhibiting albumin denaturation than compared to both the plant extracts.



Heat-induced hemolysis

The hydroalcoholic extract of V. trifolia concentration being studied for its capacity to inhibit hemolysis at 50, 100, 200, 400, 600, and 800 ug/ml concentrations has exhibited 29.45%, 40.37%, 47.41%, 58.04%, 73.69%, and 82.43% inhibition [Graph 11] while the S. nigrum extract has exhibited inhibition of 36.63%, 45.54%, 56.31%, 80.41%, 90.21%, and 103.57%, respectively [Graph 12]. The standard drug aspirin, on the other hand, has shown 100.53% membrane stabilizing capacity at 800 μg/ml. This shows that the extract of S. nigrum is as capable of inhibiting hemolysis on exposure to heat as the standard drug aspirin.




  Discussion Top


Biological response to a disrupted tissue homeostasis is called as inflammation. Exposure to foreign particles, pollutants, or microbial invaders is some of the major plausible causes that promote inflammation.[26] Although it is a defense mechanism, any reactive species liberated during the process which leads to exaggerated oxidative stress. Oxidative stress refers to excessive production of reactive oxygen species (ROS); in addition, in hypoxic conditions, reactive nitrogen species (RNS) are produced.[27] Reactive oxygen and nitrogen species initiate pro-inflammatory gene expression, thus depicting inflammation and oxidative stress to be closely related.[28],[29] Overproduction of these ROS and RNS species for a prolonged period can cause irreversible cellular structural and functional damage.[30] This makes it imperative to select agents which specifically target both oxidative stress and inflammation. Superoxide, NO, H2O2, and hydroxyl free radical are some of the radicals generated during stress and inflammation.[31]

H2O2 occurs naturally in air at low concentration. It rapidly gets decomposed to oxygen and water and also gives rise to hydroxyl radicals (OH*). The presence of hydroxyl radicals can initiate lipid peroxidation and DNA damage. The scavenging ability of the extracts could be attributed to the phenolic content in them which can donate electrons to H2O2 and in this way probably inhibit DNA damage and lipid peroxidation.

One of the most common ROS expressed during pathological conditions such as inflammation and tuberculosis is NO. To maintain normal physiological processes, one of the prerequisites is NO gas; however, its overproduction causes stress and this stress can modify protein structure, thus hampering its normal functioning. The study shows that with increase in concentration, the absorbance values are decreasing, and this could be correlated to the scavenging capacity of the extract which in turn could be due to the presence of terpenes and triterpenoids in the extracts of V. trifolia and S. Nigrum, respectively.

The phosphomolybdenum assay is a simple quantitative and economical method used to determine the total antioxidant capacity of the extract which is in turn attributed to the presence of reductones which are responsible for preventing peroxide formation which is directly proportional to the increase in absorbance. Total antioxidant capacity of the leaves of V. trifolia is same as that of ascorbic acid, i.e., equivalent to 132 mg/g while that of whole plant of S. nigrum possesses lesser total antioxidant capacity, i.e., equivalent to 68 mg/g ascorbic acid at higher concentration.

Fenton reaction is an in vitro method of generating hydroxyl radicals, and this principle can be used in the study of oxidative damage caused to their presence. The study shows that both the extracts and the standard ascorbic acid portray equal scavenging capacity.

One of the primary causes of inflammation is the denaturation of protein. The inflammatory process involves the release of series of hydrolytic enzymes and prostaglandin which are responsible for the damage of the surrounding tissues and organelles, thus causing thermal denaturation. Both the extracts have shown maximum thermal denaturation inhibition, and this probably could be the pathway of protection from thermal denaturation and thus the anti-inflammatory activity.[32] The release of hydrolytic enzymes which is the known cause for organelle damage could be prevented by the membrane stabilizing capacity of the extract which in turn could be related to the intracellular calcium content. The membrane stabilizing effect of the extracts could be a cytoprotective action happening through the alteration in calcium influx in the erythrocytes.[33] On comparison, it is seen that the hydroalcoholic extract of S. nigrum shows better potential as inhibitors of albumin denaturation and membrane stabilizing capacity when compared to V. trifolia.


  Conclusion Top


Both the hydroalcoholic extract of whole plant of S. nigrum and leaves of V. trifolia on being evaluated for their antioxidant and anti-inflammatory activity have exhibited significant activity and that their activity is seen to be dose dependent. Further, in vivo studies confirming the aforesaid claims are in pipeline.

Acknowledgment

The authors would like to the Principal, KLEUs College of Pharmacy, Vidyanagar, Hubli, for providing research facilities.

Financial support and sponsorship

This study was financially supported by the Principal, KLE University's College of Pharmacy, for research facility.

Conflicts of interest

There are no conflicts of interest.



 
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