1Research intern, Bioroot Exploration India Pvt Ltd, Thiruvananthapuram, Kerala, India.
2Founder/Managing Director, Bioroot Exploration India Pvt Ltd, Thiruvananthapuram, Kerala, India.
The present study investigates the phytochemical constitution and therapeutic potential of aqueous extract of H.indicus leaves. Hemidesmus indicus, commonly known as Indian sarsaparilla, is an Indian medicinal plant renowned for its medicinal value. It is a twining, semi-erect shrub that grows in mesophilic to semi-dry conditions at an altitude of 600m. The plant contains various bioactive compounds which accounts for its therapeutic properties. Extracts of the plant exhibits significant pharmacological properties like anti-oxidant, anti-diabetic, hepatoprotective effects, wound healing activity, anti-ulcer, anti-inflammatory, anti-arthritic, anti-microbial, and many more. Qualitative and quantitative studies indicate the presence of phytochemical compounds like alkaloids, flavonoids, tannins, saponins, phenols and terpenoids. The leaf extract showed significant anti-oxidant, anti-inflammatory and anti-arthritic activity than that of standard. The anti-diabetic and anti-ulcer activities are maximum at the lowest concentration; however, the anti-microbial activity was not found on the extract. Wound healing activity was most significant at highest concentration (500 µg/ml). The extract also portrays notable hepatoprotective effects, with the lowest concentration exhibiting greater cell viability.
Hemidesmus indicus, commonly referred to as Indian sarsaparilla is a traditional medicinal plant belonging to the family Apocynaceae [1]. The plant is a repository of numerous bioactive compounds like alkaloids, flavonoids, tannins, steroids, glycosides and phenolic compounds that are responsible for its therapeutic potential. The major pharmacological properties of the plant encompass anti-oxidant, anti-diabetic, anti-inflammatory, anti-cancerous, anti-ulcer, anti-arthritic, anti-fungal, hepatoprotectivity, neuroprotection, cardio protection, and wound healing [2,3,4]. The phytochemicals present in the plant can be extracted by soxhlet extraction using water as solvent. Oxidative stress is a major characteristic of chronic diseases that can be combated by improving the endogenous anti-oxidant defenses [2]. Phytochemicals like flavonoids, triterpenoids and phenolics present in the plant performs anti-oxidant property, thereby reduces the oxidative stress [5]. These phytochemicals are also valued for its anti-diabetic property. Diabetes is an intricate chronic metabolic disorder, caused by the impairment of regulation in blood glucose level, that leads to very serious health complications [6,7]. Treatment with the extract effectively reduces lipid peroxidation in liver and kidneys and reverses all the metabolic alterations imposed by diabetes [8]. Another therapeutic potential of the plant extract includes hepatoprotectivity. Compounds that have the potency to reduce inflammation and restore the function of liver are called as hepatoprotectants [9]. Hepatoprotectivity of H.indicus is mostly due to the presence of coumarin olignoids, such as hemidesmin-I and hemidesmin-II[10]. The plant extract also showed anti-arthritic activity. Arthritis is a complex disease caused by inflammation in the joints and are characterised by symptoms like pain, stiffness and deformities of the joints [11]. The anti-arthritic property is due to the presence of phytonutrients such as polyphenols, phenols, flavonoids and steroids present in the plant [12]. These compounds also demonstrate anti-inflammatory property. Inflammation is defined as a safeguarding response of the body against pathogens or injury [13]. Saponin, a phytonutrient in H.indicus have anti-inflammatory activity against edema induced by formalin [14]. Chronic inflammation can result in the impairment of mucosal lining of stomach that can lead to ulceration [15]. Peptic ulcer is considered as the imbalance of digestive acids and the mucosal protective layer of the stomach [16]. Extract of H.indicus have potential antiulcer activity, improving mucous secretion, thereby protecting the gastric mucosa and sub-mucosa from inflammatory reaction [17]. Antiulcer property of the plant is due to the presence of phytochemicals like alkaloids, tannins, phenols and saponins [16]. These compounds also have wound healing activity. Wound healing is a combined and complex sequence of several biochemical and cellular events [18]. The extracts of H.indicus have anti-oxidant, anti-inflammatory and anti-fungal properties which can contribute to wound healing by preventing infections, thereby stimulates wound healing activity [4,19]. Anti-fungal activity is another therapeutic application of H.indicus extract. A compound that can selectively eliminate fungal pathogens from host organism with minimal toxicity is known as an anti-fungal agent. H.indicus contain a phytonutrient called glycoside, that inhibits the adherence of microorganism to the host cell and thereby reduces its pathological effects [20]. The plant extract is potential for inhibiting the growth of fungal pathogens like Candida albicans, Aspergillus niger and Aspergillus fumigatus [21]. The study is intended to evaluate the therapeutic potential of leaf extract of H.indicus by exploring the presence of its phytochemicals, which may exhibit anti-oxidant, anti-diabetic, hepatoprotective, anti-arthritic, anti-inflammatory, anti-ulcer, wound healing properties, as well as antifungal effects.
MATERIALS AND METHODS:
Chemicals and Reagents
3-(4,5- dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (SRL), Acarbose, Acetaminophen, Aluminium chloride (NICE), Ascorbic acid (HIMEDIA), Bovine Serum Albumin (SRL), Bromocresol green (MEDILISE), Chloroform (MEDILISE), Diclofenac (SIGMAALDRICH), Dinitrosalicylic acid, Diosgenin (SRL), Dulbecco’s Modified Eagle Medium (HIMEDIA), Dulbecco’s Phosphate Buffer Saline (HIMEDIA), Egg albumin (MEDILISE), Ethanol, Ferric chloride (Kanton Laboratories), Folin – Ciocalteu (MEDILISE), Gallic acid (NICE), Gelusil, Gentamicin (HIMEDIA), Glacial Acetic acid (NICE), Hydrochloric acid (MEDILISE), Linalool, Methanol (NICE), Perchloric acid (MEDILISE), Phosphate Buffer Saline (HIMEDIA), Potassium ferrous cyanide, Potato Dextrose Agar (HIMEDIA), Quercetin (SRL),Silymarin, Sodium carbonate (NICE), Sodium hydroxide (Kanton Laboratories), Sodium potassium tartrate (MEDILISE), Sodium nitrate (Kanton Laboratories), Starch (SRL), Sulphuric acid (MEDILISE), Tannic acid (MEDILISE), Trichloroacetic acid (MEDILISE), Wagner’s reagent (Kanton Laboratories), vanillin (NICE), α-amylase (Kanton Laboratories).
Instruments
Visible spectrophotometer (Thermo SCIENTIFIC), Water bath (ROTEK), Weighing machine (SHIMADZU), Phase contrast microscope, Biosafety cabinet Type - II B II (ROTEK), Water bath (ROTEK), Autoclave (FOURTECH), CO2 Incubator (FORMA SERIES II WATER JACKET), soxhlet apparatus, Magnetic stirrer (Lab Companion).
Cell lines
L929 and HepG2 cell line (a gift from Bioroot Exploration India Pvt Ltd).
Preparation and extraction of the sample
Fresh leaves of H.indicus were collected from Thiruvananthapuram, Kerala, India. The leaves were washed, dried, powdered and transferred to a timble and placed for soxhlet extraction. 50 ml of double distilled water was added as solvent to a round bottom flask and the process was carried out at 80°C for 6 hrs. The extract was collected on the same flask and stored at 4°C for future use [22].
Qualitative screening of phytochemicals
Test for alkaloids
A few drops of Wagner’s reagent were added to 1 ml of the sample. A reddish-brown precipitate was formed, which indicates the presence of alkaloids in the test sample [23,24].
Test for phenols
A few drops of FeCl3 were added to 1 ml solution of the sample and mixed well by shaking. A bluish-black colour was formed after shaking which indicates the presence of phenols in the test sample [23,24].
Test for saponins
1ml of sample solution was mixed with 1ml distilled water and shaken vigorously. The foam formation after shaking indicated the presence of saponin in the sample [24].
Tests for flavonoids
2 ml of 2% NaOH were added to the sample and mixed well. A yellow colour was observed. A few drops of Dil.HCL were added to this mixture and the colour disappears. This showed the presence of flavonoid in the test solution [23].
Test for terpenoids
In 1ml of the sample, 2ml chloroform were added. To this mixture a few drops conc. H2SO4 were added along the side of the test tube and mixed well. The formation of a reddish-brown colour indicates the presence of terpenoids in the sample taken [24,25].
Test for glycosides
A few drops of aqueous NaOH were mixed with the sample solution. No chemical reaction occurred which indicates the absence of glycoside in the test solution.
Test for tannins
1ml of 10% NaOH were added to the sample and shaken vigorously. A slight emulsion was formed which indicates the presence of small concentration of tannin in the taken sample.
Test for coumarins
To the sample few drops of 10% NaOH and chloroform were added. No colour reaction formed, which indicates the absence of coumarin in the sample.
Test for steroids
10 ml of chloroform and conc.H2SO4 were added to the sample along the side of the test tube. No chemical reaction occurred; which confirmed that steroids are absent in the sample solution [24,26].
Quantitative estimation of phytochemicals
Alkaloids
5 mL of phosphate buffer and 5 mL of bromocresol green solution were added to 1 mg/ml of the sample. The complex was extracted by transferring the mixture into a separating funnel to which 4ml of chloroform was added by shaking vigorously and collected in a test tube. Different concentrations of quercetin from 0.2 to 1 mg/ml were taken as standard. The absorbance was read at 470 nm [27].
Phenolic compounds
To 1mg/ml of sample 2ml of FC reagent and 7.5 % sodium carbonate diluted in 4ml double distilled water were added in the ratio 1:10. The test tube was then kept at dark for incubation for about 30 mins. Different concentrations of gallic acid from 10 to 100 µg/ml were taken as standard. The absorbance was read at 760 nm using a visible spectrophotometer [28].
Saponins
To 1mg/ml of sample 250µl of the reagent containing 0.8%vanillin dissolved in 1ml ethanol were added along with 2.5ml of conc.H2SO4. The test tube was incubated for 15 mins at 60°C in a water bath and allowed to cool down at room temperature. Different concentrations of diosgenin from 20 to 100 µg/ml were taken as the standard. The absorbance was read at 544nm using a visible spectrophotometer.
Flavonoids
To 1mg/ml of sample 2ml of 5% sodium nitrite was added and kept at dark for incubation for 5 mins. 3ml of 10% AlCl3 along with 2ml of 1M NaOH was added to the test tube and again the tube was kept for incubation at dark for 5 minutes. After incubation the solution was made upto 10ml by adding distilled water and again the tube was incubated at dark for 10 mins. Different concentrations of quercetin from 200 to 1000 µg/ml were taken as the standard. The absorbance was read at 510 nm using a visible spectrophotometer [29].
Terpenoids
To 100µl of 1mg/ml of sample 150µl of reagent containing 0.055g of vanillin dissolved in 1.1ml of 5% glacial acetic and 500µl of perchloric acid were added. The test tube was then incubated in a water bath for 45 mins at 60°C, and later cooled under running tap water. After cooling 2.25 ml of glacial acetic acid were added to this mixture and the absorbance was read at 548 nm using a visible spectrophotometer. Different concentrations of linalool from 200 to 1000µg/ml were taken as standard.
Tannins
To 1mg/ml of sample 0.5ml of FC reagent and 35 % Na2CO3 diluted in 1ml of double distilled water were added in the ratio 1:1. The test tube is then kept at dark for incubation for 30 mins. Different concentrations of tannic acid from 200 to 1000 µg/ml were taken as the standard. The absorbance was read at 700 nm using a visible spectrophotometer [30].
Anti-oxidant property
Anti-oxidant property of the leaf extract is evaluated using FRAP assay. Different concentrations of the sample (25, 50, 100, 200, 500 µg/ml) were prepared in solvent. 2.5 ml of sodium phosphate buffer (pH = 6.6) and 2.5 ml of ferric cyanide were added to sample and incubated for 20 mins in a boiling water bath at 60°C. After incubation 2.5 ml of TCA was added to the solutions. An aliquot of 2 ml was transferred to a fresh test tube and 2ml of distilled water and 0.5 ml of ferric chloride was added to it. Absorbance was read at 593 nm after incubating for 10 minutes at room temperature. Ascorbic acid was taken as the standard with same concentrations as that of the extract [31,32].
Anti-diabetic property
Anti-diabetic property of the leaf extract is evaluated using α-amylase enzyme. Different concentrations of the sample (25, 50, 100, 200, 500 µg/ml) were prepared in solvent. 100 µl of sodium phosphate buffer and 100 µl of α-amylase were added to each test tubes and incubated for 10 mins at RT. It is followed by adding 1 ml of starch and again incubated for 10 minutes. Subsequently, 1 ml 3,5-Dinitrosalicylic acid (DNS reagent) was added and tubes were incubated in a boiling water bath for 10 mins. After cooling, 100 µl of distilled water was added and the absorbance was read at 540 nm using a visible spectrophotometer. Acarbose were taken as the standards at the same concentrations as that of the sample [33].
% inhibition = [(AC-AS)/AC] x 100, where AC is the absorbance of the control and AS is the absorbance of the sample.
Anti-inflammatory property
Anti-inflammatory property of the leaf extract is evaluated using protein denaturation method. Different concentrations of the sample (25, 50, 100, 200, 500 µg/ml) were prepared in solvent. 200 µl BSA and 1.8 ml PBS buffer (Ph = 6.4) were added to the sample and incubated for 20 mins at RT, followed by 5 mins of incubation in a boiling water bath at 70°C. After incubation, the tubes were cool down and absorbance was measured at 660 nm using visible spectrophotometer. Diclofenac was taken as the standard with the same concentrations as that of the sample [34].
% inhibition = [(ACAS)/AC] x 100, where AC is the absorbance of the control and AS is the absorbance of the sample.
Anti-microbial property
The anti-microbial activity of H.indicus is evaluated using agar well diffusion method. Two bacterial species, Bacillus cohnii and Staphylococcus aureus and two fungal species Aspergillus niger and Candida albicans were used for the test. The bacterial species were inoculated in a nutrient broth whereas the fungal species are inoculated in potato dextrose agar (PDA), both are incubated in an incubator for 24hrs. Nutrient agar and PDA were prepared and autoclaved as the media, and poured into sterilized petri plates, on which the respective microbes are plated after incubation. Wells were made using pipette tips and different concentrations of the sample (25, 50, 100, 200, 500 µg/ml) were poured into it. An antibiotic, gentamicin was used as the positive control and autoclaved distilled water was used as the negative control. The plates were then incubated for microbial growth.
Anti-arthritic assay
The anti-arthritic activity of H.indicus was assessed using protein denaturation method. Different concentration of the sample (25, 50, 100, 200, 500 µg/ml) were prepared and made-upto 1 ml using the solvent. 200 µl of egg albumin were added to the extract, along with 2.8 ml of PBS buffer (pH = 6.4). After mixing all the reagents, the tubes were incubated for 15 mins in an incubator and again for 5 mins in a boiling water bath at 70°C. After cooling down at room temperature, the absorbance was read at 660 nm using visible spectrophotometer. Diclofenac was taken as standard with the same concentration as that of the sample.
% inhibition = [(ACAS)/AC] x 100, where AC is the absorbance of the control and AS is the absorbance of the sample.
Wound healing property
Wound healing activity of H.indicus was determined using scratch assay. 0.3 x 106 cells/ml of L929 cell line were seeded in a 6 well plate and incubated until they reach a confluency of 80 %. The culture media was discarded after incubation and a scratch was made on each well using a sterile micropipette tip. Different concentrations of the sample (25, 50, 100, 200, 500 µg/ml) were added to the wells, after adding 1 ml of fresh media. One of the remains untreated, leaving it as the control. The wound healing property was detected by measuring the area of gap closure after a period of 24 hrs [35].
% of wound closure = (A0h – A24h)/A0h x 100 where A0h and A24h is the area of wound at 0th hour and 24th hours.
Hepatoprotective property
Hepatoprotectivity of leaf extract was detected using MTT assay against HepG2 cell line. The cells were plated on a 24 well plate and incubated until the cells reaches its maximum confluency. After incubation, 1 ml of fresh media was added after discarding the used one. The wells were treated with Acetaminophen, except for two wells, that are selected as vehicle control, and the plated were again incubated for 24 hrs. The media was replaced later with fresh media containing different concentrations of the test sample and standard, and the wells were incubated for another 24 hours. On the next day, 700 µl of MTT and 100 µl of DPBS were added to the wells along with 500 µl of fresh media after removing the old one. The plates were incubated for 3 hrs and subsequently 200µl of DMSO was aspired to the medium for dissolving the formazan crystals formed by the reduction of MTT. After 20mins the absorbance was read at 570 nm using a visible spectrophotometer. Silymarin was taken as the standard drug.
Anti-ulcer property
Anti-ulcer activity of leaf extract of H.indicus was determined using acid neutralizing capacity method. 5 ml of different concentrations of the sample (25, 50, 100, 200, 500 µg/ml) were prepared in 5 conical flask and are made upto 70 ml using distilled water. The solution was stirred for 1 min, and later for 15mins after adding 30 ml of dil HCL, using a magnetic stirrer. After stirring, 2 - 3 drops of phenolphthalein was added to the mixture, and then titrated against 0.5N NaOH until a pink colour appeared. 5 ml of Gelusil (2.5g) was taken as the standard - anti-ulcer agent.
RESULTS
Extraction of H. indicus
Fresh leaves of H.indicus were dried, powdered, and placed for soxhlet extraction, using water as solvent (Figure 1 and 2).
<a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192706-3.png" target="_blank">
<img alt="Fresh, Dried and Powdered Forms of Indicus Leaves.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192706-3.png" width="150">
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Figure 1: Fresh, Dried and Powdered Forms of Indicus Leaves
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<img alt="Extract of H. indicus leaf.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192706-2.png" width="150">
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Figure 2: Extract of H. indicus leaf
Phytochemical analysis of aqueous extract of H.indicus
Phytochemical analysis of leaf extract of H.indicus showed presence and absence of various bioactive compounds (Figure 3 and Table 1).
<a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192706-1.png" target="_blank">
<img alt="Figure 3.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192706-1.png" width="150">
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Figure 3: Qualitative Analysis of Phytochemicals (A- Terpenoids, B- Phenols, C- Alkaloids, D - Flavonoids, E- Tannins, F- Saponins, G- Steroids, H- Glucosides, I- Coumarin)
Table 1: Qualitative Analysis of Phytochemicals (‘+++’ – High Expression, ‘++’ - Moderate Expression, ‘+’ - Low Expression, ‘-’ - Absent)
|
Phytochemicals |
Results |
||
|
Alkaloids |
+ |
+ |
- |
|
Phenols |
+ |
+ |
+ |
|
Saponins |
+ |
+ |
+ |
|
Flavonoids |
+ |
+ |
+ |
|
Terpenoids |
+ |
+ |
+ |
|
Glycosides |
- |
- |
- |
|
Tannins |
+ |
- |
- |
|
Coumarins |
- |
- |
- |
|
Steroids |
- |
- |
- |
Quantitative phytochemical analysis
Alkaloids
Quantity of alkaloids from the aqueous extract of H.indicus leaves is calculated using the equation y = 0.256x + 0.882 from the standard curve of quercetin (Figure 4, Figure 5, Table 2, and Table 3).
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<img alt="4.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192706-0.png" width="150">
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Table 2: Different Concentrations and Absorbance of Quercetin
|
= |
Absorbance at 470 nm |
|
0.2 |
1.24 |
|
0.4 |
1.40 |
|
0.6 |
1.55 |
|
0.8 |
1.68 |
|
1 |
2.38 |
Table 3: Quantity of alkaloids in leaf extract
|
Phytochemical
|
Quantity
|
|
Alkaloids
|
0.998mg/ml
|
Phenols
The quantity of phenol from aqueous extract of H.indicus leaves is calculated using the equation y = 0.3155x - 0.2623 from the standard curve of gallic acid (Figure 6, Figure 7, Table 4, and Table 5).
<a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-19.png" target="_blank">
<img alt="5.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-19.png" width="150">
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Table 4: Different concentrations and absorbance of gallic acid
|
Concentrations (µg/ml) |
Absorbance at 760 nm |
|
10 |
0.016 |
|
40 |
0.393 |
|
50 |
0.692 |
|
75 |
1.060 |
Table 5: Quantity of phenolic compounds in leaf extract
|
Phytochemical
|
Quantity |
|
Phenolic compounds
|
2.8103µg/ml
|
Saponins
Quantity of saponins from the aqueous extract of H.indicus leaves is calculated using the equation y = 0.1172x + 0.0318 from the standard curve of diosgenin (Figure 8, Figure 9, Table 6, and Table 7).
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<img alt="6.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-18.png" width="150">
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Table 6: Different Concentrations and Absorbance of Diosgenin
|
Concentrations (µg/ml) |
Absorbance at 544 nm |
|
20 |
0.098 |
|
40 |
0.202 |
|
60 |
0.297 |
|
80 |
0.434 |
|
100 |
0.568 |
Table 7: Quantity Of Saponins in Leaf Extract
|
Phytochemical |
Quantity |
|
Saponins
|
3.271µg/ml
|
Flavonoids
The quantity of flavonoids from aqueous extract of H.indicus leaves is calculated using the equation y = 0.45x + 0.226 from the standard curve of quercetin (Figure 10, Figure 11, Table 8, and Table 9).
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<img alt="7.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-17.png" width="150">
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Table 8: Different Concentrations and Absorbance of Quercetin
|
Concentrations (µg/ml) |
Absorbance at 510 nm |
|
200 |
0.718 |
|
400 |
1.158 |
|
600 |
1.501 |
|
800 |
1.912 |
Table 9: Quantity Of Flavonoids in Leaf Extract
|
Phytochemical |
Quantity |
|
Flavonoids
|
0.534µg/ml
|
Terpenoids
The quantity of terpenoids from aqueous extract of H.indicus leaves is calculated using the equation y = 0.1362x + 0.1094 from the standard curve of linalool (Figure 12, Figure 13, Table 10, and Table 11).
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<img alt="8.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-16.png" width="150">
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Table 10: Different concentrations and absorbance of linalool
|
Concentrations (µg/ml) |
Absorbance at 548 nm |
|
200 |
0.276 |
|
400 |
0.335 |
|
600 |
0.548 |
|
800 |
0.613 |
Table 11: Quantity Of Terpenoids in Leaf Extract
|
Phytochemical |
Quantity |
|
Terpenoids
|
0.7898µg/ml
|
Tannins
The quantity of tannins from aqueous extract of H.indicus leaves is calculated using the equation y = 0.2011x + 0.0263 from the standard curve of tannic acid (Figure 14, Figure 15, Table 12, and Table 13).
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<img alt="9.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-15.png" width="150">
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Table 12: Different concentrations and absorbance of tannic acid
|
Concentrations (µg/ml)
|
Absorbance at 700 nm
|
|
200 |
0.223 |
|
400 |
0.431 |
|
600 |
0.636 |
|
800 |
0.828 |
Table 13: Quantity Of Tannins in Leaf Extract
|
Phytochemical |
Quantity |
|
Tannins
|
0.8683µg/ml
|
Anti-oxidant property
The anti-oxidant activity of leaf extract was assessed using FRAP assay with ascorbic acid as the standard. The result showed that, the extract has more anti-oxidant property than the standard.
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<img alt="10.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-14.png" width="150">
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Table 14: Absorbance Value of Ascorbic Acid and Sample
|
Concentrations (µg/ml) |
Absorbance at 593 nm |
|
|
|
Ascorbic acid |
Sample |
|
25 |
0.477 |
25 |
|
50 |
0.978 |
50 |
|
100 |
1.346 |
100 |
|
200 |
1.829 |
200 |
|
500 |
2.2 |
500 |
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<img alt="Absorbance of standard and sample at 593 nm.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-13.png" width="150">
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Figure 17: Absorbance of standard and sample at 593 nm
Anti-diabetic property
The anti-diabetic activity of leaf extract was evaluated using α-amylase enzyme, with acarbose as the standard. As the concentration of the standard increases, the percentage inhibition also increases, whereas the sample showed higher inhibition at lower concentrations.
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<img alt="Anti-Diabetic Property of Acarbose and Leaf Extract Of H. Indicus.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-12.png" width="150">
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Figure 18: Anti-Diabetic Property of Acarbose and Leaf Extract Of H. Indicus
Table 15: Percentage Inhibition of Acarbose and Sample
|
Concentrations (µg/ml) |
% of inhibition |
|
|
|
Acarbose |
|
|
25 |
18.793 |
25 |
|
50 |
26.476 |
50 |
|
100 |
35.619 |
100 |
|
200 |
33.079 |
200 |
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<img alt="Percentage Inhibition of Standard and Sample.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-11.png" width="150">
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Figure 19: Percentage Inhibition of Standard and Sample
Anti-inflammatory property
The anti-inflammatory activity of leaf extract was determined using protein denaturation method. The results showed that both the standard and sample exhibited higher level of inhibition at the greatest concentration.
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<img alt="Figure 20.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-10.png" width="150">
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Figure 20: Anti-Inflammatory Activity of Diclofenac And Leaf Extract Of H. Indicus
Table 16: Percentage Inhibition of Diclofenac and Sample
|
Concentrations (µg/ml) |
% of inhibition |
|
|
|
Diclofenac |
Sample |
|
25 |
9.305 |
25 |
|
50 |
20.425 |
50 |
|
100 |
23.659 |
100 |
|
200 |
29.179 |
200 |
<a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-9.png" target="_blank">
<img alt="21.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-9.png" width="150">
</a>
Figure 21: Percentage Inhibition of Standard and Sample
Anti-Microbial Property
Anti-microbial activity of leaf extract was evaluated using agar well diffusion method. The result indicates that the extract did not exhibit any anti-microbial activity towards the tested organisms. Only the positive control showed zone of inhibition against pathogenic microbes.
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<img alt="Figure 22.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-8.png" width="150">
</a>
Figure 22: A - S. aureus, B - Angier, C - C. albicans, D - B. cohnii. PC Gentamicin, NC - autoclaved distilled water
Anti-arthritic property
The anti-arthritic activity of leaf extract was determined using the protein denaturation method. The results showed that both the standard and sample exhibited higher level of inhibition at the greatest concentration.
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<img alt="Figure 23.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-7.png" width="150">
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Figure 23: Anti-Arthritic Property of Diclofenac and Leaf Extract Of H. Indicus
Table 17: Percentage Inhibition of Diclofenac and Sample
|
Concentrations (µg/ml) |
% of inhibition |
|
|
|
Diclofenac |
Sample |
|
25 |
31.851 |
25 |
|
50 |
36.298 |
50 |
|
100 |
38.341 |
100 |
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<img alt="24.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-6.png" width="150">
</a>
Figure 24: Percentage Inhibition of Standard and Sample
Wound healing property
Wound healing activity of leaf extract was determined using scratch assay by analyzing and comparing the area of gap closure between treated and untreated cells within a time period of 24 hrs.
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<img alt="Figure 25.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-5.png" width="150">
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Figure 25: Area Of Gap Closure Before and After Incubation Of 24 Hrs.
Table 18: Percentage Of Wound Closure at Different Concentrations
|
Concentrations (µg/ml) |
Percentage of wound closure |
|
0 |
14.959 |
|
25 |
10.485 |
|
50 |
7.896 |
|
100 |
8.399 |
|
200 |
9.310 |
|
500 |
17.597 |
<a href="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-4.png" target="_blank">
<img alt="Percentage Of Wound Closure.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-4.png" width="150">
</a>
Figure 26: Percentage Of Wound Closure
Hepatoprotective property
Hepatoprotectives effect of aqueous leaf extract was assessed using MTT assay against HepG2 cell line. The result showed that, the extract has more hepatoprotective effect compared to standard. Silymarin was used as the standard drug.
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<img alt="Figure 27.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-3.png" width="150">
</a>
Figure 27: HepG2 cell line after treated with different concentrations of standard and sample
Table 19: Percentage Inhibition of Standard and Sample
|
Concentrations (µg/ml) |
% of cell viability |
|
|
Standard |
Sample |
|
|
25 |
80.222 |
25 |
|
50 |
43.175 |
50 |
|
100 |
75.766 |
100 |
|
200 |
62.674 |
200 |
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<img alt="Figure 28.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-2.png" width="150">
</a>
Figure 28: Percentage cell viability of standard and sample
Anti-ulcer property
Anti-ulcer activity of leaf extract was evaluated using acid neutralizing capacity method. Gelusil was used as standard. The result showed significant anti-ulcer property for the sample.
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<img alt="Figure 29.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-1.png" width="150">
</a>
Figure 29: Acid Neutralizing Capacity of Different Concentrations of The Sample Against Gelusil
|
Concentrations (µg/ml) |
Volume of NaOH |
Acid neutralizing capacity of antacid |
|
25 |
48.00 |
0.96 |
|
50 |
46.50 |
0.46 |
|
100 |
44.30 |
0.22 |
|
200 |
45.60 |
0.11 |
|
500 |
45.00 |
0.04 |
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<img alt="Figure 30.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250501192420-0.png" width="150">
</a>
Figure 30: Acid Neutralizing Capacity of The Sample
DISCUSSION
This study aspires to examine the therapeutic potential of H.indicus, evaluating the presence of various phytochemicals, along with anti-oxidant, anti-diabetic, anti-arthritic, anti-inflammatory, hepatoprotectivity, wound healing, anti-ulcer, and anti-microbial properties of the plant. Phytochemicals present on the aqueous extract of H.indicus leaves were evaluated using various qualitative and quantitative assays. The results of the assays unveiled the presence of bioactive compounds like alkaloids, flavonoids, tannins, phenols, saponins and terpenoids, whereas compounds like glycosides, coumarin and steroids were found to be absent in the extract. (Shalini, 2021) conducted similar experiment on aqueous and alcoholic root extract of H.indicus, and the result showed the presence of steroids, flavonoids, phenolic compounds, tannins and lignins. Quantitative analytic assays were carried out for detecting the quantity of aforementioned phytochemicals present in the extract. Similar result was reported by (Balaji et al., 2017) from their experiment conducted on screening phytochemicals present on the ethanolic root extract of H.indicus. The anti-oxidant activity of H.indicus was detected using FRAP assay. The result showed that, the sample has more anti-oxidant potential compared to standard. The highest anti-oxidant activity was observed at the highest concentration (500µg/ml) of the extract. Studies conducted by (Ravikiran et al., 2016) and (Mary et al., 2003) showed similar anti-oxidant activity of root and leaf extract of H.indicus. From their experimental observations, they suggest that, H.indicus have notable free radicle scavenging activity, by neutralizing the highly charged molecules that cause oxidative stress, and thereby protecting the cells from cellular damage caused by oxidative stress. The anti-diabetic activity of H.indicus was evaluated using α-amylase inhibition assay. The leaf extract showed significant α-amylase inhibition property at variable concentrations, with the highest range of inhibition was obtained at the lowest concentration (25µg/ml) of the sample. It demonstrates that, as the concentration increases, anti-diabetic property of the extract decreases. (Taidala et al., 2024) reported that methanolic root extract of H.indicus also exhibited similar inhibition against α-amylase enzyme activity. The extract contains anti-diabetic agents that can regulate blood glucose level, regain enzymatic and metabolic processes and minimize lipid peroxidation (Gayathri and Kannabiran, 2008). Anti-inflammatory activity of H.indicus leaf extract was evaluated using protein denaturation method. The result showed up remarkable anti-inflammatory potential of the sample, compared to standard used, signifying its capability in reducing inflammation. The extract showed variable inhibition pattern, with the lowest concentration exhibiting minimal protein inhibition. Study by (Dutta et al., 1982) showed similar anti-inflammatory activity of H.indicus extract. Saponin, a phytochemical present on the extract of H.indicus attributes to its anti-inflammatory potency (Lalrinpuia et al., 2017). Anti-arthritic activity of H.indicus leaf extract was also assessed by protein denaturation method. The result suggests that, the extract have significant anti-arthritic property that increases steadily with increase in its concentration, indicating that it is beneficial for pharmacological applications, demanding persistent protein inhibition. (Abiraamasri and Lakshmi, 2016) reported similar findings from their study. (Mehta et al., 2012) described that, anti-arthritic property of H.indicus is attributed to compounds like terpenes, sterols, and phenols and polyphenols present on the extract. The anti-microbial activity of H.indicus leaf extract was examined using agar well diffusion method. Bacterial species like Bacillus cohnii and Staphylococcus aureus and fungal species such as Aspergillus niger and Candida albicans were taken for detecting the anti-microbial potential of the leaf extract. From the result it is observed that, the leaf extract of H.indicus does not exhibit any anti-microbial activity. The zone of inhibition appeared only on the positive control. The absence of anti-microbial activity by the extract may attribute to various factors such as chemical structure of the compounds, the particular strain of the organisms used or even the experimental conditions applied. Wound healing property of H.indicus leaf extract was estimated using scratch assay against L929 cell line. The outcome of the assay indicates that, greater stimulation for cell migration was performed by the extract at its higher concentration (500µg/ml). This demonstrates that, at this level of concentration, the extract can stimulate wound healing by accelerating the repair of damaged tissues, and thus, the extract can be used for the treatment of cuts, burns and other skin injuries (Ganesan et al., 2012). Experiment made by (Kurapati et al., 2012) endorsed that H.indicus carries significant wound healing property. The anti-oxidant and anti-inflammatory properties of the extract can enhance its wound healing ability by preventing infections. Hepatoprotective effect of H.indicus leaf extract was detected using MTT assay on HepG2 cell line. The result showed that, the extract has more hepatoprotective effect compared to standard. The extract at different concentrations exhibits variable hepatoprotective effects, in which the lowest concentration (25µg/ml) of the sample performs maximum hepatoprotective effect. (Baheti et al., 2017) conducted similar experiment on animal model. The study states that methanolic root extract of H.indicus express significant hepatoprotective effect against CCl4 paracetamol induced hepatic cell damage. Anti-ulcer activity of H.indicus leaf extract was determined using acid neutralizing capacity method. The result showed significant anti-ulcer property of the extract at its lower concentration (25µg/ml). The anti-ulcer activity of the extract decreases steadily with increase in its concentration. This indicates the dose depending acid neutralizing potency of H.indicus leaf extract. Study conducted by (Manonmani et al., 1995) using ethanolic extract of H.indicus showed significant hepatoprotective effect in rat models. The presence of phytochemicals like saponins, alkaloids, phenols and tannins attributes to the hepatoprotective effects of H.indicus extract (Anoop and Jegadeesan, 2002).
CONCLUSION
The present study focus on analyzing the therapeutic potential of leaf extract of H.indicus and screening of phytochemicals present in it. Qualitative phytochemical analysis authenticates the presence of bioactive compounds like alkaloids, flavonoids, saponins, phenolics, tannins and terpenoids. The study showed significant anti-oxidant, anti-diabetic, anti-inflammatory, anti-arthritic, anti-ulcer, hepatoprotective and wound healing activity. Our study also focused on in-vitro tests, that provide strong evidence for therapeutic effectiveness of H.indicus leaf extract in curing various ailments such as diabetes, inflammation, ulcers and wounds. Further studies using in-vitro models can assist in better understanding of translational potential and therapeutic mechanism of the plant. This additional research will provide important insights of biological activity of the extract and its sustainability for applying in present medical treatments.
REFERENCES
Ashna Shiju, Dr. Parvathy Prasad*, Therapeutic Potential of Hemidesmus Indicus, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 4, 55-79 https://doi.org/10.5281/zenodo.15318543
10.5281/zenodo.15318543
