Preparation And Evaluation of Herbal Gel from the Extract of Tinospora Cordifolia to Treat Inflammation

Tinospora cordifolia, commonly known as Guduchi or Giloy.It is a versatile medicinal plant long used in Ayurveda, Siddha, and Unani medicine. It contains a rich phytoconstituent like alkaloids, glycosides, steroids, flavonoids, and polysaccharides. These compounds can be studied for further pharmaceutical research [1]. These organic substances have physiological effects on the human body, interacting with pathogens to stop their growth at various phases of development and leaving the body free from disease. These bioactive compounds give the plant multiple health benefits, including boosting immunity, reducing inflammation, reducing oxidative stress, controlling diabetes, protecting the liver and brain, and showing anti-cancer effects[2].

Inflammation is a complex biological defence mechanism that often manifests with pain and involves several physiological changes. That can include increased vascular permeability, protein denaturation, and alterations in cell membranes[3]. It is triggered when body cells are injured by microorganisms, physical trauma, or chemical irritants, leading to cellular stress and the activation of an inflammatory cascade. This process is typically characterized by the classical signs of redness, heat, swelling, pain, and functional impairment at the affected site. Inflammation serves as one of the body’s primary nonspecific defence mechanisms[4]. The tissue response to a minor injury, such as a cut, follows a similar pattern to that produced by burns, radiation, or microbial infections. Its main purpose is to dilute, neutralize, or isolate harmful agents that invade the body, while initiating a sequence of biological events that promote tissue repair and healing. Common triggers of inflammation include infections, burns, physical injuries, and immune-related reactions[5].

The Topical Drug Delivery System (TDDS) is designed to deliver therapeutic agents directly to the site of action through the skin, ensuring localized drug concentrations with minimal systemic exposure. As an easily accessible organ, the skin provides an effective route for the local administration of drugs, especially for treating dermatological conditions such as acne, eczema, and psoriasis. TDDS formulations may include creams, gels, foams, sprays, and medicated patches for external use, as well as mucosal applications for internal use. The advantages of topical delivery include avoidance of first-pass metabolism, convenience, enhanced patient compliance, and targeted therapy[6]. Challenges such as skin irritation, limited permeability, and allergic responses may occur. According to the World Health Organisation (WHO), herbal medicines comprising plant derived materials or their extracts are increasingly valued for their therapeutic benefits in treating various ailments. A thorough understanding of the skin’s anatomy and physiology, which covers approximately 2 m² and constitutes 16–18% of body weight, is essential for the design of effective topical formulations. Among these, gels are semisolid preparations characterised by a smooth, solid-like consistency, widely used in topical therapy for controlled drug release and improved patient acceptance[7,8].

Classification of Inflammation:

Inflammation can be broadly categorized into three main types:

1. Acute inflammation
2. Chronic inflammation
3. Miscellaneous inflammation

MATERIALS AND METHODS:

Pre formulation study:

To ascertain novel drug substances have essential physicochemical parameters, and to ensure the safety, efficacy, and stability profile of the formulation, preformulation studies are required. During this phase of development, we studied the physicochemical characteristics and incompatibility of the pharmaceutical ingredients and how they interact with different formulation elements [9,10]

Pharmacognostic investigation:

1. Plant collection- T.cordifolia plant was collected in February 2024 from the local community market, West Bengal state, India.
2. Organoleptic Characterization: Colour, odour, taste, and size of the leaf and flower were observed [8,11].
3. Physicochemical Characterization: After botanical evaluation, the shade-dried plant material was subjected to kept for 2 weeks before undergoing the extraction process of maceration. Size reduction was done to get coarse particles, and then passed through a sieve. 20 to get uniform particles. Then, the uniform size was subjected to standardization with different parameters as per the literature [12,13].
4. Solvent selection: Choose a solvent system that is composed of 90% distilled water and 10% methanol or chloroform because this prevents oxidation and hydrolysis and contains more amounts of antioxidant moieties than other solvent systems. The solvent used for the extraction is menstruum[14].

Extraction and Storage:

Pouring: After performing the initial steps and allowing the system to settle for a while, these two were placed on a Rotary Shaker, which maintained 100 rpm and the specified strength in a closed flask stand for twenty-four hours, shaking frequently during six hours, and then stood for eighteen hours[15].

Filtration: Then the extract solution was filtrate through Whatman filter paper and a Buchner Funnel, filtrate was collected again in a 250 mL conical flask. For rapid filtration, vacuum filtration is used[15,16].

Solvent Evaporation: The filtered solvent is collected into a petri dish. These were then placed on a Hot plate for the solvent evaporation. Temperature is maintained approx 80°C until complete removal of solvent. To remove the solvent completely utilize 3 days, about 4 hours each day.

Extract Collection: The dried and solidified extract was scraped out of the Petri Plates (used during the solvent evaporation process) using a sharp Scraper.

Extractive Yields: The weight of the dried and scraped materials was measured to calculate the extractive yields, and these were then stored in a freezer for future usage. The extractive yield was measured as 0.171 g of extract of Tinospora cordifolia per gram of dry mass.

Phytochemical Screening:

Preparation of Stock Solution:

The stock solutions were prepared using 10 mg of both of the extracted dry sample and dissolving it into Methanol separately.

Test for Alkaloids:

Mayer’s reagent test: Taken 2 ml of the methanolic extract sample in test tubes. A few drops of Mayer’s reagent (potassium mercuric iodide) were added to each sample. Any change in color or precipitate (White / pale yellow) formation was observed [11,12].

Wagner’s reagent test: Taken 2 ml aqueous extract sample in test tubes. A few drops of Wagner’s reagent (2g of iodine and 6g of potassium iodide in 100 ml of distilled water) were added to each sample. Any change in color (reddish) formation was observed [11,12].

Test for saponins (frothing test):

 2ml of methanolic extract mixed with 10 ml of distilled water in test tube and shaken vigorously for 5 minutes and allowed to stand for 20 minutes. After that observe whether the honeycomb froth (foam) layer is present or not, which indicates the presence of saponin[17].

Test for Flavonoid Group:2ml of the methanolic stock solutions were taken in separate test tubes. Add prepare sodium hydroxide solution. If a yellow colour is observed, then add concentrated hydrochloric acid (HCl). Colourless sample solution indicates the presence of flavonoid groups [11,23].

Test for Phenolics (FeCl3 Test):To the methanolic extracts, a freshly prepared aqueous solution of Ferric Chloride was added and observed for any change in color.

Test for Tannins :

3 ml of methanolic extract of the sample in separate test tubes. After that, in the sample solution, 2 ml of 5% ferric chloride (FeCl3) solution was added. Any change in color or precipitate formation was observed [19].

Test for Saponin:

1-2 ml methanolic extract sample in separate test tubes. 5 ml distilled water was added to each sample. Test tubes are shaken vigorously for 5 minutes, and allowed to stand 20 minutes. After that observed the honeycomb froth (foam) layer is present or not which indicate the presence of saponin [11,20]. 

Test for Carbohydrates:

2 ml aqueous extract sample in separate test tubes. 2 ml of benedict reagent was added to each sample. Then that solution was heated in a boiling water bath for 3-5 minutes. Any change in color or precipitate formation was observed [11,21].

Test for Phenolics:

To the 3ml aqueous extracts, a freshly prepared aqueous solution of Ferric Chloride (Fecl3) was added and observed for any color change.[11,22]

Test for Flavonoid:

2 ml of the aqueous extracted sample solutions were taken in separate test tubes. Add prepared sodium hydroxide (NaOH) solution. If a yellow color is observed then add concentrated hydrochloric acid (HCl). Colorless sample solution indicate the presence of flavonoid groups [11,23].

Test for Glycoside:

Taken 1 ml of aqueous sample in a test tube. Added 5-10 ml of dilute Hydrochloric acid (HCl). Then that solution was heated in a boiling water bath for 10 minutes.  After that carbon tetrachloride and an equal amount of ammonia added. Shake well to mix properly. Sample solution color change into pink or red color indicates the positive result [11,18].

Test for Coumarins:

2 ml of water extract of the sample in separate test tubes. After that, in the sample solution added 3 ml of 10% Sodium hydroxide (NaOH) solution. The sample solution color changes into yellow color indicating the presence of coumarins [11].

Test for Quinones:

Taken 2 ml of water extract of the sample solution with 1 ml of concentrated sulphuric acid(H2SO4) solution. The sample solution color change to red indicates the presence of quinones [23] .

Test for Anthocyanins &Betacyanins :

Take 2 ml of water extract of the sample in each separate test tube. After that, in the sample solution 1 ml of 1N Sodium hydroxide (NaOH) solution. Sample solution color change into blue or bluish-brown color indicates the presence of anthocyanins and betacyanins [24].

Determination Of Total Flavonoid Group:

TFC for each extract was estimated by the protocol mentioned in the work of Bala et al. (2018 with minor changes. In plant extracts the total flavonoid content are used as determinative of antioxidant activity. The higher values of total flavonoids content suggest that it may have prevent oxidative damage and also a greater potential to scavenge free radicals .Aluminum chloride colorimetric method's fundamental tenet is that it forms acid-stable complexes with flavonoids' C-4 keto group either their C-3 or C-5 hydroxyl group. The compound is then seen in UV at 430 nm [28].

Procedure:7 mg of crude extract was melted in 1.4 mL of Methanol, and 100 µL of DMSO was added for better dissolution. This was then centrifuged at 3500 rpm ,the supernatants were collected in fresh marked Eppendorf tubes. From this 100 µL of supernatant was added to 5 different Eppendorf tubes followed by the addition of 400 µL Methanol in each.500 µL of 2% AlCl3 solution was added in each tube (0.1 g AlCl3 in distilled water of 5ml)[27,28].These were then subjected for incubation at 37°C in the incubator.The standard curve for the estimation of TFC was obtained  where Quercetin was applied as the standard flavonoid.Methanol was applied as the blank sample.At 430 nm using a UV Spectrophotometer measured the absorbance.Results were taken in triplicates and calculations were done using the linear regression equation as mentioned in the cited article and expressed value as µg of Quercetin Equivalent mg of dry extract[29].

Determination Of Total Phenol Content:

The estimation of TPC was performed calorimetrically .. In plant extracts the total phenolic content are used as determinative of antioxidant activity. The higher values of TPC suggest that it may have prevent oxidative damage and also a greater potential for scavenge free radicals. The whole process is described below stepwise. The Folin-Ciocalteu assay is a commonly used method for the evaluation of TPC in several samples. It is based on the principle that phenolic compounds can be oxidized by a mixture of Folin-Ciocalteu reagent and a reducing agent, such as sodium carbonate or sodium hydroxide, to form a blue-coloured complex and that can be measured spectrophotometrically at 765nm, an increase in the absorbance would signify the higher presence of phenolic in the sample[30].

Process: 5 mg/ml of extracts were taken in Eppendorf tubes and added 100 µL DMSO for better dissolving companied by addition of 1400 µL distilled water.Then 5 min. centrifuged at 3500 rpm .Resulting supernatants were collected in separate Eppendorf tubes. 0.5 ml of each supernatant of the extracts was taken in test tubes and 2 ml of Folin- Ciocalteu Reagent (FC Reagent) was added to each of these (the FC reagent was diluted with distilled water in a ratio of 1:10).This was then neutralized with the addition of 4 ml of 7.5% Na₂CO₃ solution (prepared fresh by dissolving 7.5 g Na2CO3 in 100 mL distilled water). These were then incubated for 30 minutes at room temperature.Gallic Acid was applied as the standard material. A stock solution of Gallic Acid was made by dissolving 10 mg of Gallic Acid in 10 ml of distilled water (stock concentration was 1 mg/mL).For the preparation of the standard curve, amounts of Gallic acid ranging from 25 µg, 50 µg, 75 µg, … to 150 µg were taken and were allowed to react with the solution containing FC reagent as mentioned above. At 765 nm using a UV Spectrophotometer for measured absorbance with a blank prepared with distilled water only[30]. The experiment was performed in triplicates (n=3). Gallic Acid standard curve was prepared and TPC for the extracts were calculated using standard equation obtained through linear regression. Results were given as µg of Gallic Acid Equivalent· mg of dry extract[31].

DPPH Activity AssaY:

The procedure for the DPPH activity assay was obtained from Djarkasi et al., 2019 with slight modification.DPPH assay’s principle emerge on the ability of DPPH, a stable free radical, to accept an electron or hydrogen radical, undergo color change.DPPH is a deep purple-colored compounds in its radical form. When it reacts with an antioxidant compound or reducing agent, such as flavonoids, alkaloids, or other organic compounds which results in the DPPH reduction[32]. This reduction leads to a decolorization of DPPH solution, changing its color from purple to yellow. The delocalization also gives rise to the deep violet colour with an absorption in the ethanolic solution at around 517 nm. When DPPH solution mixing with a substance that donate a hydrogen atom, the reduced form is produced and violet colour is lost[31,33]. DPPH assay is widely used as a quick and simple method to evaluate the radical scavenging action of natural products, synthetic antioxidants, and plant extract.

Preparation of 0.395mg DPPH Solution:For the preparation of DPPH solution 0.394mg of DPPH solution  was dissolved in 100ml of methanol in a small glass beaker and stirred well until a deep violet colour was obtained. These were then wrapped in aluminum foil as DPPH is very-sensitive.

Preparation of Ascorbic Acid Stock Solution[32]:10mg of Ascorbic acid was weighed carefully and mixed with 100ml methanol as a solvent and 100µL DMSO was also added for better solubility.A 1 mg per ml extract’s stock solution was prepared using methanol as a solvent and 100 µL DMSO was also added for better solubility.The concentrations used for the test samples ranged between 50µg,100µg,150µg…up to 200 µg, made the volume up to 2 mL using methanol.The wrapped DPPH was taken out and 1 ml was pipette out for the addition in every sample.1 ml of this freshly prepared DPPH solution was added to each test tubes containing test sample and were incubated in dark for about 30 minutes in room temperature.Ascorbic acid is used here as the standard antioxidant sample. A stock solution was prepared using distilled water (1 mg per ml), then amounts were taken as 50µg,100µg, … to 200µg. The rest of the additions are same as mentioned above.A positive control was prepared without the test sample in the solution.A blank was prepared which contained 4 ml of Methanol only.With time, a change in the colour of the solutions were visible which turned from violet to golden and then eventually very light golden .At 517 nm, absorbance was measured in UV Spectrophotometer. Triplicates results were taken.% Inhibition values were determined from the absorbances of both standard and test materials which were later used to determine the IC50 values by plotting a graph of % Inhibition vs Concentration and determining the unknown using GraphPad Prism software.

%I = [(A Pos Ctrl – A Sample)/A Pos Ctrl] × 100 …eq.1

1. 1. %I: Percentage Inhibition
   2. A Pos Ctrl: Absorbance of Positive Control
   3. ASample: Absorbance of Sample

Preparation of 10 % Neutral Buffered Formalin:

Neutral buffered formalin (NBF) 10% which is used general histological fixative purpose and widely employed for preparing samples for light microscopy. To prepare 10 % NBF, this procedure mentioned in Tristan. M 1967 with minor changes.

Procedure For 1000 ml 10 % NBF: Mix 100 ml formalin with 900 ml water.Then maintain the PH 6.8-7.2 by adding the buffers .After got the correct PH stored in a well closed container until usage.

Herbal Gel Preparation:

 Two gelling agents were utilized in the formulation process at two different concentrations Azadirachta indica 20%;Tagetes erecta 50%; Ageratina adenophora 30%, resulting in the manufacture of two distinct batches of gels. Both Xanthan gum and Carbopol 934 were used as gelling agents in this instance. 

Preparation of gel with Carbopol 934: accurately measured four grams of carbopol 934 and mixed it with 50 ml of purified water. After setting the beaker aside to allow the carbopol to expand for 30 minutes, stir the mixture with a mechanical or lab stirrer set at 450 rpm for 20 minutes. Add extract aqueous solution approx. 20 ml then slowly pour 2 ml of glycerol mix and fill it with weighed liquid paraffin, stirring thoroughly. After all of the carbopol had dispersed then added the remaining distilled water, and drop by drop triethanolamine was added to the formulations to balance pH (4.8–6) and get the gel to the desired consistency.

Preparation of gel with Xanthan gum: A beaker containing 3 g of Xanthan gum was filled with 50 ml of distilled water. Set aside the beaker to let the Xanthan gum swell for ten minutes and then stirring should be done continuously to prevent flocs formation. Take 5 ml of propylene glycol and the small amount of extract into a separate beaker and thoroughly mix in the propyl and methylparaben. Then add glycerine as a preservative. Mix it properly and allow standing 6 hours to get semi-solid concentration.

Evaluation and standardization of gel:

1. PH: Using a digital pH meter, the formulation's conductivity and pH were measured in millivolts (mV). The glass electrode was calibrated using the equipment-specific solutions (pH of 4.00 and 7.00). The preparation was allowed to reach equilibrium while being measured for approximately five minutes. Average values were computed when the formula's conductivity and pH were analyzed in triplicate [9].
2. Appearance and Homogeneity: A visual examination was used to assess the generated individual and polyherbal gels for homogeneity and physical appearance(color, odor, etc) [25].
3. Viscosity: Using a Brookfield viscometer, the prepared herbal gels' viscosity was determined. Spindle No. 6 is used to rotate the gel at 100 revolutions per minute.^[17]
4. Spreadability: Spreading 0.5 g of the gel over a circle between two horizontal planes with a 2 cm diameter pre-marked on a glass plate allowed for the measurement of the gel's spreadability. A second glass plate was then used. Five minutes were allowed for a half-kilogram of weight to lie on the upper glass plate. After the gel was spread, the circle's diameter was measured.[26]
5. Extrudability: Standard capped collapsible aluminum tubes were filled with gel compositions, and the ends were sealed with a crimp. The tube weights were noted. The tubes were clamped after being positioned between two glass slides. After covering the slides with 500 grams, the cap was taken off. Weighing was done on the amount of extruded gel that was collected. The extruded gel's percentage was determined as follows: >80% extrudability is good, >90% extrudability is excellent, and >70% extrudability is fair [25] .
6. Stability study: the gel stability investigations are conducted at 37°C for 3 months.[27]

Table 1: Macroscopical Evaluation of Tinospora cordifolia

Table 2: Physiochemical Analysis of Tinospora cordifolia Aqueous Extract

Anti-Inflammatory Study:

All in vivo experiments will be conducted in accordance with applicable national regulations and institutional guidelines for animal care and use. Experimental protocols will be reviewed and approved by the Institutional Animal Ethics Committee (IAEC) before initiation .Humane endpoints minimization of pain/distress, and appropriate euthanasia criteria will be specified in the approved protocol.

Due to Restrictions of CPCSEA for the use of animals in experimental pharmacological research, such as ethical issues and the lack of rationale for their use when other suitable methods are available. The Tinospora cordifolia extract exhibited a marked in vitro antioxidant and potential anti-inflammatory effect, as evidenced by its high phytochemical content and radical-scavenging capacity. The extract showed appreciable total phenolic content (TPC) and total flavonoid content (TFC), confirming the presence of bioactive polyphenols and flavonoids that contribute to its pharmacological activity. In the DPPH free-radical-scavenging assay, the extract demonstrated a strong, concentration-dependent inhibition of DPPH radicals, with a low IC?? value indicating high antioxidant potential comparable to that of the standard antioxidant (ascorbic acid). Similarly, the hydrogen-peroxide scavenging assay revealed significant ability of the extract to neutralize H?O? radicals, further supporting its role in preventing oxidative damage that triggers inflammatory responses. A positive correlation was observed between the TPC/TFC levels and radical-scavenging activity, suggesting that polyphenolic and flavonoid compounds are the primary contributors to the antioxidant efficacy of T. cordifolia. The overall results confirm that the extract effectively scavenges both stable and reactive oxygen species, reducing oxidative stress that leads to inflammation. These findings validate the traditional use of Tinospora cordifolia in treating inflammatory conditions and provide biochemical evidence supporting its potential as a natural anti-inflammatory agent. The study therefore highlights T. cordifolia as a promising candidate for further mechanistic and in vivo investigations to develop safe, plant-based anti-inflammatory therapeutics.

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RESULT:

Total Flavonoid Content (TFC):

• The standard equation presented there was y= 0.0054x + 0.1122 …. (1) with a R2 value of 0.9917
• The values of ‘x’ was determined from the equation for the test samples, using their average absorbance values as the ‘y.’
• Then it was multiplied with the concentration present in the total volume test sample and the value was calculated for 1 mg/ml extract and showed as µg QE.gm of the dry extract.
• The amount considered for plotting this graph range 25µg, 50µg, ….150µg.
• The linear regression was drawn marked with bold blue line that represented the standard equation.
• The experimental data is represented at a table in below.

Table 3: Determination of Total Flavonoid Content of Tinospora cordifolia

Table 4: Standard Calibration Curve of Quercetin Solution

Table 5: Total Flavonoid Content of Tinospora cordifolia

The TFC values are represented as a column in the graph below , made using Graphpaf Prism 8.02 software.

The values for TFC are represented as the mean ±SD, the TFC values of Tinospora cordifolia extract was found to be 62.9 ± 1.05357 mg QE/gm of dry extract.The ‘X’ values obtained through the standard equations were multiplied with the amount of sample present in the solution.Then the values were calculated for 1gm of extract and represented as their respective QE equivalent.

Total Phenol Content (TPC):

Gallic Acid standard:

To obtain a standard equation, the experiment was performed on a 3day basis (n=3) with the standard phenolic compound Gallic Acid.

The Standard equation obtained was [y= 0.0067x+0.0325] …. (2) with a R2 value of 0.9932

The values of ‘x’ was determined from the equation for the test samples, using their average absorbance values as the ‘y.’

Then it was multiplied with the concentration present in the total volume test sample and the value was calculated for 1 mg extract and showed as µg GAE· mg of dry extract-1.

The amounts considered for plotting this graph ranges from 25 µg, 50 µg, 75 µg,100µg and 150 µg.A linear regression was drawn marked with the bold line that represents the standard equation.

Result Of Test Samples: The values calculated TPCs of Tinospora cordifolia has been reported in the table below.

Table 7:  TPC values of Test Samples

The mean ± SD for Acorus calamus roots extract was 21.8904 ± 3.6264 mg GAE/gm of dry mass.

The ‘x’ values obtained through the standard equations were multiplied with the amount of sample present in the solution.

Then then value was calculated for 1 mg of extract and represented as their respective GAE equivalent.

DPPH Activity Assay [ % Inhibition]:

Ascorbic Acid Standard:

• A standard graph of the % inhibition of DPPH by Ascorbic Acid was plotted using different concentrations.
• The % inhibition were calculated from the absorbance values of 4 different concentration.
• The maximum % inhibition was recorded at 70µg/mL of Ascorbic Acid for 0.4 mM DPPH.
• The mean ±SD values of the % inhibition for each concentration is as follows, 50µg/mL=47.080±6.817,100µg/mL=50.173±5.784,150µg/mL=52.349±6.473, 200µg/mL=57.492±2.089.
• The absorbance values for each concentration of Ascorbic Acid are provided in a tabular from in next page along with the % inhibitions.

Table 9: % Inhibition Values of Ascorbic Acid in DPPH

% Inhibitions of Test Samples:

Tinospora cordifolia:

The % inhibition values of Tinospora cordifolia extract is assigned by mean ± SD and follows according to concentration, 50 µg/mL= 28.129±4.108,

100µg/ml=50.598±3.330,150µg/ml=45.086±4.790,200µg/ml=55.842±2.058

The summery of the % inhibition values calculated through absorbance data is shared below for test samples.

The IC50 values calculated with the help of these % inhibition data are showed in the coming page.

The values of % inhibition is utilized to form a group a comparison of the individual concentrations of the test along with the standard substance all in one which is shared just below. A two-way ANOVA is performed for the analysis of the variance in this case.

The statistical measured are mention within the graph itself. All the % inhibition measures have been mentioned on previous pages.

The highest % inhibition has been recorded for the test samples at 200µg/ml mark which is 55.842±2.058.

Significance differences were found when the Acorus calamus extract compared with Ascorbic acid and the significance levels are mentions in the plot above.

The IC50 value calculations are being mentioned below.

Ascorbic Acid Standard:

The IC50 values were interpolated using concentrations up to 70 µg/mL for Ascorbic Acid. That is because the highest % inhibition was reached there and further than that the graph was no longer linear.

The values of IC50 were 98.488 µg/ml in 0.4 mM DPPH solution.

The IC50 of the Acorus Calamus root extract in DPPH was measured using interpolation of the unknown value in the linear regression.

The value of IC50 was 157.707 µg/ml in 0.395mM DPPH solution.

All the graphs and tables are presented in below.

H₂O₂ Scavenging Activity Assay [ % Inhibition]:

Ascorbic acid standard:

A standard graph of the % inhibition of H₂0₂ by Ascorbic Acid was plotted using different concentration. The % inhibitions were calculated from the absorbance values of four different concentrations. The maximum % inhibition was recorded at 70µg/mL of AA for 43mM H₂0₂. The mean ±SD values of the % inhibition for each concentration is as follow,50µg/mL=47.080±6.817,100µg/mL=50.173±5.784,150µg/mL=52.349±6.473, 200µg/mL=57.492±2.089. The absorbance values for each concentration of Ascorbic Acid are provided in tabular form at below with the % inhibition.

% Inhibition of Test samples:

Tinospora cordifolia:

The % inhibition values of Tinospora cordifolia extract is assigned by mean ±SD and follows according to concentrations 50µg/ml=45.4037±3.196,100µg/ml=50.521±2.910,150µg/ml=45.724±6.470,200µg/ml=56.558±3.377.The summary of the % inhibition values calculated through absorbance data is shared below for the test samples.

The IC50 values calculated with the help of these % inhibition data are showed in the coming pages.

The values of % inhibitions are utilized to form a group a comparison of the individual concentrations of the test along with the standard substance all in one which is shared just below. A two-way ANOVA is performed for the analysis of the variance in this case.

The statistical measured are mentioned within the graph itself. All % inhibition measures have been mentioned on previous pages. The highest % inhibition has been recorded for test sample at 200 µg/ml which is = 56.558±3.377. Significant differences were found when the Ascorbic Acid was compared with Tinospora cordifolia extract and the significance levels are mentioned in the plot above. The IC50 value calculation are being mentioned below.

H₂O₂ Scavenging Activity Assay [IC50 Calculation]:

Ascorbic Acid Standard:

The IC50 Values were interpolated using concentrations up to 120µg/mL for Ascorbic Acid. This is because the highest % inhibition was reached there and further than that the graph was no longer linear. The mean ± SD value of IC50 was 98.488 µg/ml in 43 mM H₂O₂ solution.

Tinospora cordifolia Extract:

The IC50 of the Tinospora cordifolia roots extract in H₂O₂ was measured using interpolation of the unknown value in the linear regression. The value of IC50 was 132.879 µg/ml of 0.1462ml of H₂O₂ solution. All the graphs and tables are presented at below.