Comprehensive Phytochemical, Anti-Oxidant and GC-MS Analysis of Strobilanthes jomyi P. Biju, Josekutty, Rekha & J.R.I.Wood

MATERIALS AND METHODS

Study area: The plants were collected from Pandi, Kasaragod District of Kerala, India (latitude-12.528705, longitude-75.213831) in December, 2020. The plants were then raised in the greenhouse inside the university throughout this study from January to August, 2021. The plants are still maintained for future studies and analysis.

Identification of plant material: The plant was authenticated and identified by Biju. P. Assistant Professor, Department of Botany, Government College Kasaragod, Kerala. The voucher specimens (Voucher No. FRLH-123445) were deposited at FRLHT Bengaluru.

Preparation of plant extract: Fresh leaves, stems and roots of Strobilanthes jomyi were cleaned with tap water followed by distilled water. The cleaned parts were shade dried at room temperature. Then the dried samples were powered by a mechanical grinder and stored in an airtight container⁸.

Methanolic extraction: Shade-dried leaves, stems and roots of S. jomyi were extracted with methanolic solvents by Soxhlet apparatus for 9 hrs. The solvents were vaporized using a rotary evaporator under a vacuum at 37°C. Later the extracted plant materials were stored in sterile falcon tubes in cold conditions for future studies⁸. In qualitative analysis, 2 g of dried samples were dissolved in 20 mL of methanol and sieved through Whatman No. 1 filter paper. The final extract was used for further qualitative tests⁹.

Physicochemical characters
Total ash value: A cleaned silica crucible was heated for about half an hour in an electric muffins furnace until it became red hot and cooled in a desiccator. The empty weight of the silica crucible was then taken. A total of 3 g of leaves, stem and root powders were taken separately in the silica crucible and kept in an electric muffins furnace (R.V Lab Care Technologies, Karnataka, Bengaluru, India) at 600-625°C for 2 hrs. Once the powdered plant material was turned to carbon-free ash, the crucible was then kept in a desiccator to cool and the weight of the carbon-free ash was noted down to find out the total ash content¹⁰.

Acid insoluble ash: The carbon-free ash acquired from the total ash was boiled with 25 mL of 2 M hydrochloric acid for about 5 min. Then insoluble ash was filtered using ashless filter paper, which was then cleaned with regular hot water to remove the traces of acid content and ignited in electric muffins furnace at 450°C for 1 hr. Finally, the crucible was cooled in a desiccator (Borosil, Mumbai, India). The weight of the sample was taken to obtain the percentage of acid-insoluble ash¹⁰.

Water-soluble ash: The ash acquired from the total ash was heated with 25 mL of distilled water with continuous stirring for 5 min. The filtrate was then transferred through ashless filter paper and ignited in electric muffins furnace at 450°C for 1 hr. Crucible was then cooled and weighed the sample to obtain the percentage of water-soluble ash¹¹.

Determination of moisture content: A total of 0.5 g of powdered sample was heated at 105°C till all the moisture content was removed. The powdered materials were then placed in a Shimadzu-unibloc moisture analyzer (Shimadzu Corporation, Kyoto, Japan) to analyze the sample’s moisture content¹².

Mineral assay using atomic absorption spectroscopy: About 0.5 g of plant sample was mixed with 2 mL of 65% HNO₃ and 30% of 1M H₂O₂ and incubated overnight at room temperature. The samples underwent an acid digestion process at 220°C for 5 min using Whatman No. 1 filter paper. Already made up (distilled water) samples were then filtered and determined the mineral content using Shimadzu atomic spectrometer-AA-6880 (Shimadzu Corporation, Kyoto, Japan)¹³.

Qualitative estimation of phytochemical analysis
Detection of alkaloid
Dragendorff’s Test: Almost 3 mL of crude extract was mixed with 1 mL of dilute hydrochloric acid followed by 1-2 mL of Dragendorff’s reagent. Orange-brown precipitation indicated the presence of alkaloids¹⁴.

Detection of flavonoid
Ammonia Test: A total of 5 mL of diluted ammonia solution was added with 3 mL of extract and 1 mL of concentrated H₂SO₄. Yellow colure indicated the presence of flavonoids¹⁵.

Detection of saponin: The amount of 1 mL of plant extract was mixed with 2 mL of distilled water and a few mL of sodium bicarbonate solution. The appearance of stable honeycomb froth after vigorous shaking indicated the presence of saponins¹⁶.

Detection of tannin: A total amount of 0.5 mL of 5% lead acetate solution was treated with 2 mL of plant extract. The white colour revealed the presence of tannins¹⁷.

Detection of terpenoids: Detection of terpenoids with slight modification. A total of 5 mg of plant extract was mixed with 5 mL of methanol followed by 2 mL of chloroform. The whole solution was then warmed and cooled. About 1 mL of the concentrated H₂SO₄ was added to the test tube. The reddish-brown colour in the interface revealed the existence of terpenoids¹⁷.

Detection of steroids: About 1 mL of chloroform was treated with 1 mL of extract, followed by concentrated H₂SO_4. The appearance of brownish colour shows the presence of steroids¹⁸.

Detection of resins
Turbidity Test: In 1 g of dried plant power, 10 mL of distilled water was added and ultrasonicated for 15 min at 30. The mixture was then filtered, the formation of turbidity indicated the existence of resin content¹⁹.

Detection of phlobatannin: A few drops of 1% HCl were added with 2 mL of plant extract. The formation of red precipitation reveals the existence of phlobatanni⁸.

Detection of carbohydrate
Benedict’s Test: About 1 mL of test solution was suspended with 1 mL of Benedict reagent and boiled for 2-3 min. Red, green or yellow colour denotes the existence of reducing sugar²⁰.

Detection of protein and amino acids: A total of 2 mL of extract was mixed with 1-2 drops of freshly prepared Ninhydrin reagent and heated for 2-3 min. The development of blue colour denotes the presence of amino acids, proteins, or peptides²⁰.

Quantitative estimation of phytochemical analysis
Total phenol estimation: Total Phenolic estimation was determined by 96 well-microtiter plates using Folin-Ciocalteu Method with slight changes. Maximum 25 μL of leaves, stem and root extracts of (1 mg mL^–1) S. jomyi were diluted with 75 μL of water and mixed with 25 μL of 1:1 diluted Folin-Ciocalteu Reagent. Later 96 -well microplate was shaken for about 1 min and allowed to stand for 4 min. The 100 μL of 5% (w/v) Na₂CO₃ solution was also added after 4 min of incubation and then shaken for about 1 min at a constant speed. After 2 hrs of incubation, absorbance was taken at 700 nm. The results were expressed as mg GAE/g²¹.

Total flavonoid estimation: The total flavonoid content of S. jomyi leaves, stem and roots was estimated by an aluminium chloride assay with minor changes. About 50 μL of extract were treated with 150 μL of 100% methanol, followed by 10 μL of 10% aluminium chloride and 10 μL of 1M sodium acetate. At room temperature, the reaction mixture was kept in dark conditions for 40 min and absorbance was taken at 415 nm (using a microtiter plate). The results were expressed as mg QE/g²¹.

Total carbohydrate estimation: Estimation of the total carbohydrate content of roots, stem and leaves of S. jomyi was done by the phenol-sulphuric acid method with minor modification. About 0.1 g of crushed fresh plant material was taken in a boiling tube and treated with 2.5 N of 5 mL HCl. The boiling tube was incubated in the water bath at 100°C and cooled to room temperature. Sodium carbonate was then added until saturation point is obtained. Distilled water was used to make up the volume to 25 mL. Then the reaction mixture was centrifuged for 10 min at 10000 rpm. A total of 100 μL of supernatant was taken and treated with 100 μL of 5% phenol, followed by 500 μL of 96% H₂SO₄. The test tubes were then kept in a water bath for 20-30 min and vortexed for 10 min. Absorbance was taken at 490 nm. D-Glucose was taken as a standard and expressed as mg g^–1 of fresh weight of the samples²².

Total protein estimation: The total Protein Content of S. jomyi was estimated by Lowry’s Method with slight modification. A total of 0.5 g of fresh leaf, stem and root of S. jomyi were ground with 10 mL of buffer solution (0.2 M of NaH₂PO₄ and 0.2 M of Na₂HPO₄ at a pH of 7). After centrifugation, the supernatant was collected. The 100 μL of the sample was treated with 1000 μL Reagent C (50 mL of Reagent A was prepared using 2% Na₂CO₃ was added to 0.1 N of NaOH and 1 mL of Reagent B was prepared using 1% Potassium sodium tartrate was added with 0.5% CuSO₄). The entire mixture was vortexed and allowed to stand for 10 min. The reaction mixture was incubated in dark after adding 100 μL of 1:1 Folin-Ciocalteu Reagent (Reagent D). Using a microtiter plate, the absorbance was read at 660 nm. Bovine Serum Albumin was taken as standard for the assay and results were expressed as mg g^–1 of fresh weight of the samples²².

Total proline estimation: In the experiment, 1 g of fresh sample (fresh leaves, stem and roots, respectively) was ground with 10 mL of sulphosalicylic acid (3%) and the filtrate was taken for further use. About 2 mL of the filtrate was allowed to react with 2 mL of glacial acetic acid and freshly prepared acid Ninhydrin. It was then kept in a water bath at 100°C for 1 hr. The tubes were placed in an ice bath to arrest the reaction process. To the reaction mixture, 4 mL of toluene was poured and stirred for 30 sec. In the microtiter plate, the coloured toluene layer was taken and absorbance was read at 520 nm. Standard was taken as proline and results were then expressed as mg g^–1 of fresh weight of the samples²².

Total chlorophyll estimation: The total chlorophyll of S. jomyi (fresh leaves, stem and root) was determined by the acetone method²². The test solution was prepared using 1 g of the fresh sample which was pulverized with 20 mL of 80% acetone. The mixture was centrifuged for 5 min at 5000 rpm. The supernatant was separated from the pellet. The same procedure was continued 2-3 times until the pellet became colourless with 80% acetone. The volume of supernatant was made up to 30 mL. Absorbance was taken at 645,652 and 663 nm, respectively, by using a microtiter plate. About 80% acetone was used as a blank²². Results were then expressed as mg g^–1 of fresh weight of the samples²²:

Determination of anti-oxidant activities: The antioxidant activity of the plant extracts was estimated by non-enzymatic and enzymatic methods like DPPH (2,2 -diphenyl-1-picrylhydrazyl), phosphomolybdate assay, Ferric reducing power assay and metal chelating assay.

Determination of antioxidant activity by DPPH radical scavenging assay: Estimation of antioxidant activity of the leaves, stem and root methanolic extract of S. jomyi was estimated by the revised DPPH (2,2 -diphenyl-1-picrylhydrazyl) procedure. About 500 μL of methanolic plant extract was made up of 2.5 mL of methanol followed by 1 mL of 0.004% (0.002 g in 100 mL of methanol) DPPH solution and kept in dark condition for 30 min. The wavelength was read at 517 nm by using a UV-VIS spectrophotometer-1800 (Shimadzu Corporation, Kyoto, Japan). Methanol was taken as a blank and ascorbic acid was used as standard²³. The antioxidant activity was calculated as²³:

where, X_control and X_sample are the absorbance of control without plant extract and sample with plant extract, respectively.

Determination of antioxidant activity by phosphomolybdate assay: The antioxidant activity was estimated by the Phosphomolybdate assay. To 0.1 mL of plant extracts, 1 mL of phosphomolybdate reagent was added and incubated for about 90 min in a water bath at 90°C. The whole test solution was then cooled at room temperature and absorbance was measured at 695 nm using a UV-VIS spectrophotometer. About 0.1 mL of methanol and 1 mL of phosphomolybdate reagent mixture were taken as a blank followed by ascorbic acid taken as standard. Equal volumes of 28 mM Na₃PO₄, 4 mM (NH₄)₂MoO₄ and 0.6 M H₂SO₄ were used for the preparation of Phosphomolybdate reagent²³.

Determination of antioxidant activity by (FRAP) ferric reducing power assay: The antioxidant activity was estimated by FRAP assay. A total of 1 mL of methanolic extracts of leaves, stem and root were suspended with 2.5 mL of phosphate buffer (0.2 M at pH of 6.5), followed by 2.5 mL of 1% ferricyanide. The final reaction mixture was then enveloped with foil paper and kept in a water bath for 20 min at 50°C. After 20 min, 2.5 mL of 10% TCA (trichloroacetic acid) was

added and centrifuged at 3000 rpm for 10 min. Later 2.5 mL of supernatant was collected and suspended with 2.5 mL of distilled water, followed by 0.5 mL of FeCl₃. The wavelength was measured at 700 nm by using a UV-VIS spectrophotometer. Methanol was taken as blank and ascorbic acid as standard²³.

Determination of antioxidant activity by metal chelating assay: The non-enzymatic anti-oxidant assay was performed by metal chelating assay with slight modification. A total of 1 mL of methanolic plant extract was mixed with 1 mL of 0.1 mM FeSO₄ followed by 2 mL of 0.25 mM of ferrozine. After 10 min of incubation, absorbance was measured at 562 nm using a UV-VIS spectrophotometer. Methanol was taken as standard and in addition to methanol, a remaining reagent was used as control²⁴:

where, Z_control and Z_sample are the absorbance of control without plant extract and sample with plant extract, respectively.

Gas chromatography-mass spectrometry analysis
Extract preparation: Extract prepared for GC-MS analysis with slight modification. As 1 g of powdered samples of leaves, stem and roots of S. jomyi was immersed with 10 mL of chloroform and kept for three days for incubation. The extract was filtered and evaporated at dryness. After evaporation, the solid extracts were transferred to vials and mixed with 0.5 mL of chloroform, respectively²⁵.

Gas chromatography and mass spectrometry analyses of the chloroform of S. jomyi were carried out in the Shimadzu GC-MSQP2010SE system. The mass spectrometer consisted low polarity phase: Crossbond^™ Silarylene Phase 1,4 bis (dimethylsiloxysil) phenylene dimethyl polysiloxane with ID:025 mm and 0.25 μDF, length 30 m Column. The column temperature was maintained at a maximum of 280°C throughout the analysis and the helium gas flow rate was at 1.25 mL/min. The ionization voltage was at 70 eV. The mass spectrum was obtained in the range of 1.5-1000 m/z. The peaks of GC-MS compounds in plant extracts were compared with the standard MS (NIST 2017) data library.

Statistical analysis: The statistical analysis of the present study was analyzed by SPSS statistics software version 22. The results were interpreted by One-way Analysis of Variance (ANOVA) to verify the significance by using Tukey’s Test by the probability less than p<0.05.

RESULTS

Physicochemical characters: The results of physicochemical characteristics like acid-insoluble ash, water-soluble ash, total ash value and moisture content were represented in Table 1. The total ash values and water-soluble ash values decreased in the order stem>leaves>root. Similarly, acid-insoluble ash values were higher in leaves, then stem, followed by root. The moisture content of the samples was observed to be higher in leaves than in roots followed by the stem.

Mineral assay using atomic absorption spectroscopy: Micro and Microelements of vegetative parts like stem, root and leaves of S. jomyi were shown in Table 2. The Ca content was observed to be higher in concentration in different vegetative parts of S. jomyi. The number of minerals was in the order Mg>Fe>Zn>Mn>Cu>Pb>Cd, respectively.

Qualitative phytochemical analysis: The preliminary qualitative phytochemical study of S. jomyi in different vegetative parts of methanolic extracts was presented in Table 3. The methanolic extract of leaves exhibited the existence of saponins, tannins, alkaloids, steroids, phlobatannin, carbohydrates, flavonoids, proteins, amino acids, terpenoids and resins tested negative in the methanolic extract of leaves. Similarly, qualitative estimation of methanolic stem extract also revealed the presence of tannins, flavonoids, carbohydrates, alkaloids, proteins, saponins and amino acids. The root extract of methanolic showed the existence of alkaloids, flavonoids, saponins, carbohydrates, proteins and amino acids. In contrast, resins showed a negative result in methanolic extracts of leaves, stems and roots.

Quantitative phytochemical analysis: The total phenolic, flavonoid, carbohydrate, protein, chlorophyll and proline Content of the methanolic extract of leaves, stem and root of S. jomyi were shown in Table 4-6. The studies revealed that leaves (87.43±0.44 mg GAE/g) contain more phenols than stem (5.89±1.77 mg GAE/g) and root (73.33±1.17 mg GAE/g). Similarly, flavonoid content was also observed to be higher in leaves (66.23±0.530 mg QE/ g), followed by root (12.02±0.30 mg QE/g) and stem (12.02±0.30 mg QE/g). Total carbohydrate content in fresh samples of leaves (44.75±1.28) expressed a higher value than root (36.48±0.98) and stem (24.82±2.27). Moreover, protein analysis revealed a higher quantity in leaves (17.78±0.76 mg g^–1 of fresh weight) compared to the root (10.38±0.26 mg g^–1 of fresh weight) and stem (5.15±0.34 mg g^–1 of fresh weight). The statistically significant difference in total phenol, flavonoid and carbohydrate-protein content was observed between leaves, root and stem. Similarly, the statistically significant difference in total proline content was observed to be higher in leaves (46.83±0.15 mg g^–1 of fresh weight) followed by the stem (21.15±2.47 mg g^–1 of fresh weight) and root (13.68852±0.00 mg g^–1 of fresh weight). And even the same trend was observed in total chlorophyll content in a different part of S. jomyi in leaves (2.76±0.02 mg g^–1 of fresh weight), then Stem (0.20±0.001 mg g^–1 of fresh weight) followed by root (0.04±0.006 mg g^–1 of fresh weight).

Anti-oxidant activity: The anti-oxidant assay in different vegetative parts of S. jomyi was estimated by DPPH, ferric reducing power assay, phosphomolybdate method and metal chelating assay (Table 7). The methanolic extract of S. jomyi leaves (83.64±0.46%) showed a higher percentage of anti-oxidant activities by DPPH assay than root (77.70±0.46%) and stem (21.26±0.17). Although industrially available the ascorbic acid standard showed higher anti-oxidant activity (96.31±0.001%) than plant samples. Anti-oxidant activity by ferric reducing power assay showed higher anti-oxidant activity in standard ascorbic acid (0.82±0.007 nm) by comparing with the samples like leaves, stem and root. Leaves (0.39±0.004 nm) showed higher values then followed by root (0.22±0.002 nm) and stem (0.09±0.001 nm). Phosphomolybdate assay also showed higher anti-oxidant activity in leaves (180.28±1.95 μg mg^–1 of extract) than root (150.90±6.84 μg mg^–1 of extract) and stem (27.17±5.95 μg mg^–1 of extract). Non-enzymatic assay like metal chelating assay revealed higher anti-oxidant potential in leaves (64.78±0.40%) then root (42.20±0.32%) and stem (33.70±0.32%). Statistical analysis of different anti-oxidant assays revealed significant differences between leaves, roots and then the stem.

Gas chromatography-mass spectrometry analysis: The volatile secondary metabolite present in the root, stem and leaves of S. jomyi was identified by GC-MS analysis (Table 8). The chromatograms of each vegetative part of the chloroform extract were expressed in Fig. 1-3. Bioactive compounds like 2,4-di-tert-butylphenol, phytol, squalene, neophytadiene, chloromethyl 5-chloroundecanoate and Lupeol were the most common secondary metabolites present in the chloroform extract. The retention time, molecular weight, molecular formula, percentage of area and compound name of secondary metabolites present in S. jomyi were presented in Table 6.

Fig. 1:

GC-MS chromatogram of Strobilanthes jomyi chloroform leaves extract

Fig. 2:

GC-MS chromatogram of Strobilanthes jomyi chloroform stem extract

Fig. 3:

GC-MS chromatogram of Strobilanthes jomyi chloroform root extract

DISCUSSION

The present study focused on the qualitative and quantitative profiles of the physicochemical characters, micro and macro elements, phytochemicals, anti-oxidants and GC-MS analyses of the leaves, stem and root of Strobilanthes jomyi.

Ash values were used to detect purity, quality of impurities, adulteration and authenticity of the samples. The water-soluble ash values express the major inorganic component present in the sample. Estimation of the acid-insoluble ash value was used to determine the amount of silica. The minimal amount of these three parameters represents a smaller number of inorganic substances and silica²⁶. The current study proved that the total ash value of S. jomyi stem showed the highest percentage followed by leaves and roots. Compared with the available data on Strobilanthes ciliatus total ash value of stem showed a higher value (15.4%) and leaves (12.3 %) which were a much lesser ash value than the S. jomyi. The water-soluble ash value of S. ciliatus were 6.1% for leaves and 8.2% for the stem. Similarly, the acid insoluble ash value was 2.2% for leaves and 2.4% for the stem in S. ciliatus⁷. The previous studies on Acanthus montanus (Nees) T. Anderson leaves which also belongs to the Acanthaceae family disclosed an abundant amount of moisture content compared to S. jomyi leaves²⁷. Higher moisture content leads to higher enzymatic activity, which initiates the disintegration of specific phytochemical substances¹³.

Mineral assay of S. jomyi revealed the presence of micro and macro elements. Calcium was higher in concentration and the least was cadmium. Another Acanthaceae member Andrographis echioides (L.) Nees. also revealed a higher concentration of calcium (2203 mg/100 g) and the least of Ag (0.01 mg/100 g)²⁸. However, S. jomyi showed a good amount of these elements than A. echioides. Elements like calcium improve the development of bones and contraction of muscles and help with the transmission of nerve impulses²⁹.

The Mg can turn on enzymes and control various concomitants such as blood pressure, glycemic control, myocardial contraction, etc.³⁰. It can also help in the synthesis of hemoglobin and myoglobin³¹. The Zn is another essential element for DNA synthesis and carbohydrate metabolism¹³. The Cu has a significant role in the development and growth of bones and medullary sheath in the nervous system²⁹. The Mn plays a vital role in blood clotting and bone and tissue growth³². Heavy metals like Pb and Cd are highly toxic to the human body and categorized as non-essential elements³³. Because of a good amount of micro and macro elements observed in the S. jomyi, vegetative parts can be used in various forms for the deficiency of any elements in the human body.

The qualitative phytochemical analysis is mainly dealing with the presence and absence of phytochemicals. In contrast, quantitative phytochemical analysis is mainly concerned with the quantity and concentration of phytochemicals in the plant. The qualitative phytochemical analysis of S. jomyi leaves, stem and root revealed the presence of alkaloids, flavonoids, tannins, carbohydrates, proteins and amino acids. Similarly, phytochemical studies on S. ciliates callus revealed the existence of glycosides, saponin, alkaloids, tannins, flavonoids, steroids, terpenoids and phenols in methanolic extract. Whereas ethanolic extract tested negative for glycosides and saponins¹. A phytochemical test for an ethanolic extract of S. ciliates leaf and stem also tested positive for alkaloids, carbohydrates, glycosides, phytosterols, terpenoids, flavonoids and tannins⁷. Recent studies on methanolic extract of the root of Strobilanthes heyneanus found the existence of phytochemicals like flavonoids, phenols, terpenoids, saponins, tannins, alkaloids, glycosides and carbohydrates. The report on quantitative phytochemical analysis of Strobilanthes species is limited. The available data on S. heyneanus showed the highest phenolic (126.53±2.29 μg mg^–1 of GAE of extract) and flavonoid (32.79±0.62 μg mg^–1 of QE of extract) content in its roots than S. jomyi¹⁷. The report on Strobilanthes crispus leaves also revealed the presence of TPC and TFC content in a different solvent, which was much lesser than the S. jomyi leaves of methanolic extract³⁴. Protein and carbohydrate content in different Acanthaceae members showed remarkable variation up to the species level³⁵.

Based on the phytochemical analysis, a higher quantity of phenols and flavonoids can be responsible for the antioxidant activity of the plant. Some of the best naturally occurring anti-oxidant compounds contain vitamin C, vitamin A, carotenoids and vitamin E, which showed a higher amount of anti-oxidant activity³⁵. Results proved that the vegetative parts of S. jomyi possess anti-oxidant activity in all the enzymatic and non-enzymatic assays. The previous report on the percentage of anti-oxidant activity of S. heyneanus methanolic root extract showed the highest amount of anti-oxidant activity in all the assays¹⁷. Similarly, S. crispus leaves revealed the highest percentage of anti-oxidant activity of ethanolic extract ie 5.44±1.76 μmoL L^–1 in DPPH assay and 12085.7±0.006 for mmoL g^–1 for FRAP assay³⁶. Correspondingly S. kunthiana methanolic and ethanolic extract revealed the highest percentage of radical scavenging activities¹. Ferric-reducing power assay is determined based on the absorbance value. Higher absorbance indicates higher antioxidant activity. Earlier studies have reported that anti-oxidant activities of S. heyneanus by ferric reducing power assay using methanolic extract of root showed much more anti-oxidant than S. jomyi root extract¹⁷. Among anti-oxidant studies of all the Strobilanthes genus concluded that S. jomyi also exhibits a good percentage of anti-oxidant activity in all the vegetative parts. From this study by comparing all four assays, the DPPH assay showed the highest anti-oxidant activity and is an effective protocol for determining anti-oxidant study. In the current scenario, an anti-oxidant study has a vital role in its health benefits. Anti-oxidants are comprised of a large number of chemical compounds that prevent oxidative damage caused by the free radicals in the body³⁵.

S. jomyi chloroform extract revealed the presence of various medicinally important bioactive compounds by GC-MS analysis. The available report on S. kunthiana and S. crispus also revealed secondary metabolites like S. jomyi^1,37. Bioactive compounds like 2,4-di-tert-butylphenol have properties like anti-oxidant, insecticidal, nematicidal, antibacterial and cytotoxic activities against HeLa cell lines³⁸. Phytol is another aromatic compound with antinociceptive, anti-oxidant, anti-allergic, anti-microbial and anti-inflammatory activities³⁹. Squalene is a naturally present polyprenyl compound known for its properties like cholesterol synthesis, which also has anti-oxidant and anti-cancerous potential⁴⁰. Lupeol is one of the significant components of S. jomyi. Lupeol is a pentacyclic triterpenoid with numerous anti-microbial, antiprotozoal, anti-cancerous, anti-diabetic, cardioprotective, anti-inflammatory, hepatoprotective, skin-protective and nephroprotective activities^41,42. The present study is the first report on S. jomyi. The application of the plant is highly diverse, having immense medicinal values based on photochemical, antioxidant and GC-MS studies. The prospects of this plant find wide applications in the pharmaceutical industry and also can be used in drug development in the future. S. jomyi is a storehouse of several medicinal properties that still haven’t been explored yet. It is highly recommended that this plant be looked at as a potential contributor to the medical field.

CONCLUSION

The present study on S. jomyi revealed a higher amount of phenols, flavonoids, carbohydrates, proteins, proline and chlorophyll in leaves than in roots and stems. The antioxidant activity was also higher in leaves followed by root and stem. This study concluded that S. jomyi can act as a source for many secondary metabolites that benefit humans. The current study claimed that lupeol was one of the significant compounds, which is responsible for many properties like anti-cancerous, cardioprotective, hepatoprotective, anti-microbial, etc. This particular plant can be used for drug development in the future medical field.

SIGNIFICANCE STATEMENT

This study provides immense information on phytochemical, anti-oxidant and GC-MS analysis of various vegetative parts of Strobilanthes jomyi. The current study helps future researchers to acquire knowledge on the medicinally important species of the Strobilanthes genus and provide insight into drug development and various preparation of ayurvedic formulations based on the quality of different plant parts by different experimental setups.

ACKNOWLEDGMENTS

The authors are grateful to Christ (Deemed to be University) management for providing the necessary facilities. We are also thankful to Biju P., Assistant Professor of Botany at Kasaragod Government College, Kerala and FRLHT Herbarium Collection Centre, Bengaluru, for identifying the specimen.