Phenolic, Flavonoid And Tannin Dynamics Coupled With Free Radical Quenching Ability In Leaf And Stem Of Mesosphaerum Suaveolens (L.) Kuntze

The global shift toward natural pharmacotherapy has intensified the search for plant-derived bioactive compounds capable of mitigating oxidative stress a precursor to numerous chronic pathologies including cancer, diabetes, and neurodegenerative disorders. Among the vast botanical repertoire, the family Lamiaceae stands out for its high concentration of polyphenols and essential oils. Mesosphaerum suaveolens (L.) Kuntez (synonym Hyptis suaveolens), commonly known as American Mint Weed or Vilayati Tulsi, is an aromatic, quadrangular-stemmed herb that has emerged as a prolific source of secondary metabolites with significant pharmacological potential. (Mishra et al., 2021).

The physical structure of M. suaveolens is defined by its vertical, pubescent, and lignified adult stems, which can grow to heights of 6 feet, alongside heart-shaped, toothed leaves. These leaves produce a characteristic scent attributed to chemical components such as alpha-caryophyllene and eucalyptol (Pandey and Shrisvastava, 2014). Historically, the plant has been prized for its anti-fertility, anti-rheumatic, and antispasmodic effects.

The species' adaptability is demonstrated by its dimorphic seeds, which maintain the ability to germinate under varying environmental conditions. This robustness has enabled it to establish itself as a widespread weed across Central America, Africa, and India.

While the qualitative phytochemical makeup of M. suaveolens is largely uniform globally—regularly exhibiting alkaloids, flavonoids, tannins, and glycosides—quantitative data show marked disparities. For instance, Riddhi et al. (2025) identified a total phenolic content (TPC) of 5.3 mg/ml in leaf samples alongside elevated flavonoid levels, yet Obiahu et al. (2025) recorded a TPC of 10.30 mg/100 g. These variances emphasize how external conditions, including climate and soil chemistry, modulate the plant's secondary metabolic processes. (Akharaiyi et al., 2023)

The antioxidant potential of M. suaveolens has been substantiated through various analytical methods, specifically DPPH and ABTS assays. Existing literature suggests that extraction techniques play a critical role in determining potency; methanolic leaf extracts, for example, have exhibited IC50 values ranging from 0.28 mg/ml (Soumahoro et al., 2023) to as low as 14.04 μg/m (Gavani and Prakash, 2008) l. Despite the extensive scrutiny of the leaves, there remains a notable deficiency in comparative research concerning the stem components. While certain investigations have identified the presence of proteins, starch, and tannins in the stem (Dhamale et al., 2023), a comprehensive side-by-side quantitative assessment of the antioxidant capacity between the leaf and stem represents a significant research gap.

The microenvironment frequently modulates the chemical profiles of invasive species. In particular, the canal areas of Gandhinagar constitute a distinct ecological region where anthropogenic variables and specific soil moisture conditions potentially affect the sequestration of secondary metabolites.

Research Objective: This investigation is designed to address current gaps in literature through a comparative assessment of the antioxidant and phytochemical profiles of M. suaveolens stems and leaves, specifically those sourced from the canal regions of Gandhinagar. Through the application of rigorous quantification and extraction protocols, the study endeavours to substantiate the therapeutic viability of this common weed, establishing a scientific framework for its integration into advanced medicinal formulations.

MATERIAL AND METHODS

Sample Collection

Mature plant was collected from Road side plantations of Koba Gandhinagar; India The plant was collected in winter season in mid of January. The plant was authenticated from sources like e Flora of Gandhinagar, e Flora of India and Gujrat university.

Sample Preparation

The plant material was initially washed 2 to 3 times by distilled water. Leaves and Stem were separated afterwards, using a paper towel excess water was removed and the material was kept to shade dry for 4 days. Stem was kept till 7 days due to excess water content. The leaves were directly grinded by a mechanical grinder but the stem was broken down into piece using mortar and pastel due to their large size and flexibility. Leaf powder was obtained directly. Again the exception stands with the stem, after grinding it multiple times the powder was subjected to a fine sieve to get fine powder and the leftovers was grinded again.

Extraction

The powdered plant material was extracted by macerating it in Ethanol and Methanol. 200ml of Ethanol and Methanol was added in 10 gram of plant material. The beaker was covered with a foil paper to prevent evaporation and it was kept inside a cupboard for 3 days in room temperature without disturbance. After 3 days a setup containing funnel, filter paper, test tube stands and a petri dish was built for filtering out the extract. Solution was poured into petri dish via this setup till half full then kept at room temperature till it dries out. Yield was obtained by repeating the process. Extract obtained by this process is called Cold Extract and this method is called cold extraction method.

Qualitative Phytochemical analysis

Alkaloid: Dragendroff’s Test extract up to 1ml + Few drops of Reagent alongside the test tube which leads to formation of Reddish Brown ppt. (De Silva, G. O., et al., 2017; Kumar, M. K., et al., 2011).

Tannin:  Ferric Chloride test extract 1ml+ 2ml Ferric chloride (5%) formation of bluish black colour formed            (Audu, S. A. et al., 2007; Sayma et al., 2025)

Glycoside: Keller-Killiani test. Extract 1ml+ 3ml Glacial acetic acid+ few drops of Ferric Chloride (0.5%)+ Few drops of concentrated H2SO4alongside the test tube. Brown ring at the junction and the acetic acid layer appears bluish (Singh and Kumar, 2017)

Flavonoids: Lead Acetate test extract up to 1ml+ Lead acetate leads to formation of yellow ppt (Singh and Kumar, 2017).

Phenol: Ferric Chloride test 1ml Extract+ 5% Ferric chloride few drops  Dark green or bluish black colour (Raaman N, 2006; Tiwari et al., 2011)

Saponins: Persistent Foam Test. Extract up to 1 ml and add 5ml of distilled water and shake it.

Terpenoids: Salkonski’s Test. Filtrate + Few drops of H2SO4 concentrated Golden yellow colour layer (Shaikh, J. R., et al., 2020).

Protein: Millon’s Test. 2ml extract and add Millon’s reagent whit ppt will form (Silva et al., 2017)

Carbohydrate (Starch): Iodine test. Plant extract than few drops of iodine blue black colour will appear (Rathod et al., 2022).

Quantitative Analysis

Formula for Total Yield

Total Yield percentage calculation.

Yield%=(Weight of Dry Extract)(Weight of Plant Powder Taken)×100

Total Phenol Content (TPC)

Phenol is a group of compounds and usually present in almost every plant in a good amount. To determine Total Phenol Content, I used Folin-Catechu Reagent technique. 1ml Plant extract/Gallic acid solution (1mg/ml in distilled water). Than make series of gallic from 10µl to 50µl add distilled water to make it up to 1ml. Now in a test tube the put 0.5ml Folin-Catechu (Prepared in 1:1 ratio with distilled water). Add 10ml distilled water. Now make 20% Sodium Carbonate solution and add 1ml directly after adding distilled water. Again add Distilled water till the solution is of 25ml. Now incubate it in dark for 30 minutes. Observe its Optical density at 765nm (Madhu et al., 2016)

Calculation

C=X×VM

Cà Total Phenol content

Xà  Concentration of Phenolic (mg/ml)

Và Volume taken

Mà Plant powder per ml

Now formula to find concentration of Phenol

y = mx+c

x= y-c/m

yà Absorbance of sample

mà Slope of Calibration curve

cà Concentration of Phenol (Siddiqui et al., 2017)

Total Flavonoid Content

Total Flavonoid Content (TFC)

Flavonoids are obtained from phenol. this phytochemical some time possess antioxidant potential. To determine the Total Flavonoid Content, I used Aluminium Chloride Colorimetric method. It starts with adding 1ml plant extract/ Quercetin solution (1mg/ml in methanol). Then make the series of Quercetin from 50µl to 500µl add distilled water up to 1ml and then add 4ml of distilled water. Then add 0.3ml of 55 Sodium nitrate incubate it in dark for 5 minutes. After 5 minute add 0.3ml of 10% Aluminium Chloride. At 6^th minute add 2ml of 1M Sodium Hydroxide. At last add distilled water to make total solution up to 10ml (Madhu et al., 2016). Observe its optical density at 510nm.

Calculation

C=X×VM

Cà Total Flavonoid content

xà  Concentration of Flavonoids(mg/ml)

Và Volume taken

Mà Plant powder per ml

Now formula to find concentration of Flavonoids

y = mx+c

x=y-cM

yà Absorbance of sample

Mà Slope of Calibration curve

cà Concentration of Flavonoid (Siddiqui et al., 2017)

Total Tannin Content (TTC)

These phytochemical is renowned for protecting the plant in its juvenile stage by producing very strong taste. To calculate total tannin content, I used Potassium Iodate method. It starts from adding 1 ml of plant extract/ Tannic Acid solution (1mg/ml in distilled water) in test tube. Make a series of tannic acid from 100µl to 500µl and add distilled water to make 1ml. Prepare a cold water bath and keep those test tubes in it for 5minutes. Then add 5 ml of 2.5% KIO3.  Take out the test tube and incubate it at room temperature for 25-30 minutes. Observe its optical density at 550nm (Willis, R.B, 1998)

Calculation

C=X×VM

Cà Total Tannin content

xà  Concentration of Tannin(mg/ml)

Và Volume taken

Mà Plant powder per ml

Now formula to find concentration of Tannin

y = mx+c

x=y-cM

yà Absorbance of sample

mà Slope of Calibration curve

cà Concentration of Tannin (Siddiqui et al., 2017)

Antioxidant essay (DPPH)

This is a popular and common method used to check the presence of free radical scavenging activity of a given plant material. 2, 2-diphenyl-1-picrylhydrazyl (DPPH) is a dark crystalline powder having stable free radical molecules. 4mg (0.004%) of DPPH was dissolved in 100 ml methanol. 3ml of prepared DPPH was added to various concentration of sample (200, 400, 600, 800, and 1000 µg/ml) and incubate for 30 minutes at room temperature in dark, the absorbance was read at 517nm in duplicates for each concentration in spectrophotometer. To prepare the standard make the ascorbic acid solution in 0.2mg/ml than add methanol in series of 200µl to 800µl. The DPPH antioxidant assay is based on the ability of DPPH, a stable free radical, to decolorize in the presence of antioxidants, thus a low value corresponds to a good scavenging ability. Ascorbic acid solution of same concentration is used as standard for the free radical scavenging assay. The antioxidant activity is given terms of mg/g Ascorbic acid equivalent was used as standard (He and Venant 2004; Sayma et al., 2025).

Calculation

I%=(A Control-A Sample)A Control×100

For IC_50, Take y=50

 y = mx+c

x=y-cM

yà Absorbance of sample

mà Slope of Calibration curve

cà Concentration of Ascorbic acid

Result and Discussion

Qualitative analysis result

Table 1

(Present= +, Absent= -, Highly Present= ++)

Quantitative analysis result