Determination of Antioxidant Potential of Stem, Root and Callus Extract of Caralluma stalagmifera C.E.C. Fisch

Medicinal plants are widely recognized as potential sources of natural antioxidants, which can neutralize free radicals and thereby prevent oxidative stress–related disorders such as diabetes, cancer, atherosclerosis and cardiovascular diseases. (Kishore, et al. (2010;Habibuddin et al., 2008; Marwah et al., 2006).

In the present investigation, the antioxidant potential of Caralluma stalagmifera stem, root, and callus extracts was evaluated in vitro using the DPPH free radical scavenging assay.(Gnanashree, et al,2018 Rajesh, B. et al. (2013). Rai, . et al. (2016). Kumari, et al..2018).

Caralluma stalagmiferaC.E.C. Fisch. belongs to the family Apocynaceae, comprises nearly 200 genera and over 2500 species worldwide. In India, around 13 species and 7 varieties of Caralluma are recorded of which 11 are restricted to South India (Karuppusamy & Pulliah, 2007).(Sankaran et al., 2017) Caralluma stalagmifera is  endemic to South India, bearing small dark-purple flowers and fleshy, leafless stems. Traditionally, C. stalagmifera has been employed in the treatment of diabetes, rheumatism, leprosy, fever, gastric problems, snake and scorpion bite and infections (Al-harbi et al., 1994; Tatiya et al., 2010; Bushnak et al., 2021). It exhibits various pharmacological properties such as antidiabetic, anti-inflammatory, antimicrobial, anticancer, and antioxidant activities (Bader et al., 2003;  due to the presence of bioactive constituents like pregnane glycosides, saponins, flavonoids, triterpenes, and steroidal glycosides.(Veerabhadraiah  et  al 2024;Venkatesh et ,al,2012;Rana,  et al. (2020).Rao, et al . 2012; Chaudhary & Singh, 2015)

Plants rich in phenols, flavonoids and steroidal saponins are of increasing demand as they are known to possess antioxidant compounds that can scavenge free radicals and play an important role in preventing diseases.; Although several synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoulene (BHT) are commercially available, but are quite unsafe and their toxicity is a problem of concern. (Erel, 2004;). Hence, there is an increasing demand for plant-derived antioxidants.

In the present study, evaluation of antioxidant activity using methanolic extracts  Raju et al., 2011).of stem, root, and callus extracts of C. Stalagmifera were assessed using DPPH free radical scavenging assay (Singleton et al., 1999; Nenadis et al., 2007;S). The findings showed variation in activity among different plant parts supporting its possible application in nutraceutical and pharmaceutical industries (Lanchhana et al., 2023; Deep et al., 2023; Wani et al., 2023; Ansari, et al,.2005.).

MATERIALS AND METHODS 

The C. stalagmifera plants were collected from Medikonda village from Jogulamba Gadwal District, the southern part of Telangana state in India during the month of June-July in the year 2021. The plant was authenticated by the Department of Botany of Osmania University.

Drying & Extraction:

Preparation of extraction stem and root were collected from the field growing plants of C. stalagmifera. The plant parts thoroughly cleaned and cut into pieces, dried and powdered by mechanical grinder. The 50 gram powder sample was extracted with methanol   by Soxhlet apparatus at 66-80 °Cfor  analysis of presence of different phytochemicals.  

Preparation of callus extraction:

Callus was initiated from the nodal explants of C. stalgmifera on MS medium in combination with 2.4-D (2, 4-Dichlorophenoxy acetic acid).  Callus was induced from nodal explants within 10 days of inoculation Two monthold callus was collected, dried and extraction with Methanol inSoxhlet  apparatus. antioxidant activity of methanol extract of in vitro callus was performed to know the important chemical compounds present in the callus.

Preparation of Extracts:

The powdered samples of stem, root and callus were subjected to extraction using methanol as solvent. Extraction was performed using Soxhlet apparatus and the filtrates were concentrated to dryness. The dried extracts were preserved in airtight containers for evaluation of antioxidant activity.

DPPH Free Radical Scavenging Assay:

The antioxidant activity of methanolic extracts of stem, root, and callus was determined using DPPH free radical scavenging method. A stock solution of DPPH (0.3mM) was prepared in methanol and protected from light. Different concentrations of the extracts (15–200 µL) were prepared in DMSO. For each test, 150 µL of DPPH solution was added to 3 mL of methanol containing the extract, mixed well and incubated in the dark for 15 min. Absorbance was recorded at 516 nm using methanol as blank. Ascorbic acid served as the positive control.

The percentage of radical scavenging activity was calculated using the formula:

% Antioxidant activity =

(Control Absorbance – Sample Absorbance x 100
Control Absorbance

RESULTS AND DISCUSSION

In the present study, a comparative analysis of the stem, root, and callus methanolic extracts of Caralluma stalagmifera was carried for evaluating the antioxidant potentia evaluated by  DPPH assay.. The radical scavenging activity of the extracts was compared with that of the standard antioxidant, ascorbic acid.

1. Antioxidant Activity of Stem Extract:

Methanolic stem extract of C. stalagmifera displayed dose-dependent scavenging activity across the tested concentrations (15–200 µg/ml). The percentage inhibition increased gradually from 54.23% at15 µg/ml to 78.48% at 200 µg/ml. At intermediate concentrations, the activity was 62.87% (50 µg/ml) and 68.48% (100 µg/ml). The IC?? value of the stem extract was 98.6 µg/ml suggesting moderate antioxidant potential when compared with ascorbic acid (IC?? < 50 µg/ml).

2. Antioxidant Activity of Root Extract:

The methanolic root extract also exhibited strong scavenging potential, with inhibition values ranging from 58.47% at 15 µg/ml to 84.69% at 200 µg/ml. At 50 and 100 µg/ml, the activity recorded was 67.12% and 73.89%, respectively. The IC?? value of the root extract was 89.60 µg/ml, which is lower than that of the stem extract indicating that the root extract is relatively more efficient in free radical quenching.

3. Antioxidant Activity of Callus Extract:

The in vitro–derived callus extract demonstrated comparatively higher scavenging efficiency among the three extracts. The activity increased from 15 µg/ml (63.87% ) to 200 µg/ml (87.12%) with intermediate concentrations showing 69.47% (50 µg/ml) and 76.87% (100 µg/ml). The IC?? value was 66.75 µg/ml, which was significantly lower than that of stem and root extracts highlighting the promising antioxidant capacity of callus cultures.

The antioxidant activity of all extracts followed a clear dose-dependent pattern. Among them, the callus extract exhibited the strongest radical scavenging activity (lowest IC??), followed by the root extract and then the stem extract. Although ascorbic acid showed consistently higher scavenging potential across all concentrations, the results suggest that C. stalagmifera extracts, particularly from callus tissue, are effective natural antioxidants. The superior performance of callus cultures also indicates that in vitro propagation systems can be exploited for the sustainable production of antioxidant-rich bioactive compounds, thereby reducing dependence on wild plant populations.

Table 1. Antioxidant activity of Caralluma stalagmifera stem,root and callus extract by DPPH assay.

Fig1. Percentage radical scavenging activity of Caralluma stalagmifera stem, root and callus extract.