Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 13, Number 2, October 2024 | Pages: 617-625 | DOI: 10.14421/biomedich.2024.132.617-625 ISSN 2540-9328 (online) Exploring the Therapeutic Potential of Kalanchoe Pinnata: A Comprehensive Analysis of Bioactive Compounds and Pharmacological Activities Dakshina Bhusal1, Manila Poudel2,3, Ashika Tamang1, Sujan Dhital1, Nirmal Parajuli1, Timila Shrestha1,2, Samjhana Bharati1,2, Binita Maharjan1,2, Jhashanath Adhikari Subin4, Bishnu Prasad Marasini5, Ram Lal Swagat Shrestha1,2,* 1Department of Chemistry, Amrit Campus, Tribhuvan University, Lainchaur, Kathmandu 44600, Nepal. 2Kathmandu Valley College, Syuchatar Bridge, Kalanki, Kathmandu 44600, Nepal. 3Department of Biotechnology, National College, Tribhuvan University, Lainchaur, Kathmandu 44600, Nepal. 4Bioinformatics and Cheminformatics Division, Scientific Research and Training Nepal P. Ltd, Bhaktapur 44800, Nepal. 5Nepal Health Research Council, Ramshah Path, Kathmandu 44600, Nepal. Corresponding author* swagatstha@gmail.com Abstract Natural products are vital in drug discovery for their bioactive compounds. This study evaluated Kalanchoe pinnata's leaf extracts for antidiabetic, antimicrobial, antioxidant, and cytotoxic properties. GC-MS identified seven components in hexane and twenty in methanol extracts. The ethyl acetate extract had high phenolic (485.17 mg GAE/g) and flavonoid (40.25 mg QE/g) contents. Methanol extract showed strong antioxidant (IC50: 293.53 µg/mL) and anti-alpha glucosidase (IC50: 195.39 µg/mL) activity. Antimicrobial tests showed varied effectiveness, while chloroform and acetone extracts exhibited notable cytotoxicity. ADMET predictions assessed absorption and blood-brain barrier penetration, highlighting K. pinnata's potential for drug development. Keywords: ADMET; Antioxidant; Cytotoxicity; GC-MS; Phytochemical screening. INTRODUCTION Natural products, sourced from living organisms like plants, animals, and microorganisms, constitute a wide array of bioactive compounds. They play a crucial role in drug discovery due to their abundant availability and varied chemical compositions, serving as the primary source of many medicinal active ingredients (Harvey, 2008). Typically, they possess biological functions suitable for application in drug discovery and design (Zhang et al., 2013). The medicinal properties of plants arise from their constituents, which induce physiological responses in the human body (Olowa & Nuñeza, 2013). These constituents include alkaloids, essential oils, tannins, and resins. Kalanchoe pinnata (Linn.) Pers. is a plant primarily located in temperate and tropical areas worldwide. This tall perennial has thick, fleshy, scalloped leaves and hollow stems bearing pendulous bell-shaped flowers (Okwu & Nnamdi, 2011). It has long been recognized for its diverse pharmacological properties, often utilized in traditional medicine to address various significant human ailments (Rajsekhar et al., 2016). The compounds found in the leaves of K. pinnata comprise various substances such as flavonoids, alkaloids, triterpenes, glycosides, and steroids. They are known for their potential to reduce inflammation and regulate blood sugar levels, and are widely used in traditional remedies for diabetes management, kidney stones, and fighting infections (Ojewole, 2005). Kidney stones, a prevalent urological condition, involve the development and occasional passage of clusters of crystals within the urinary tract (Singh & Saini, 2009). Compounds in K. pinnata hinder kidney stone formation and offer diverse benefits: quercetin reduces crystal deposition, phenolics dissolve stones, kampferol inhibits crystal formation, glycosides protect kidneys, and steroids aid in stone passage, collectively providing comprehensive management for kidney stones and related urinary issues (Nagpal & Sharma, 2020). Diabetes Mellitus (DM) represents a significant multifaceted chronic ailment contributing substantially to global morbidity (Arroyave et al., 2020). Traditional clinical approaches to managing DM have primarily revolved around administering oral hypoglycemic medications and insulin (George et al., 2019). Many of the therapy choices in current use are burdened by Manuscript received: 30 August, 2024. Revision accepted: 09 January, 2025. Published: 23 January, 2025. https://doi.org/10.14421/biomedich.2024.132.617-625 618 Biology, Medicine, & Natural Product Chemistry 13 (2), 2024: 617-625 various adverse effects. These encompass issues like weight gain and hypoglycemia associated with insulin, beta-cell exhaustion with secretagogues, lactic acidosis with sensitizers like metformin, and gastric disturbances with alpha-glucosidase inhibitors. Extracts from K. pinnata have shown efficacy in controlling blood sugar levels (Efanova et al., 1998; Kimmel & Inzucchi, 2005; Lalau & Race, 1999; Patil et al., 2013). Besides these, plant extracts also possess the ability to inhibit the growth of certain harmful microorganisms. They can hinder germ proliferation and their antimicrobial potency can be evaluated accordingly (Kalemba & Kunicka, 2005). The main objective of the research was to investigate the biological effects such as antidiabetic, antimicrobial, antioxidant, and cytotoxic properties of various extracts obtained from K. pinnata leaves, as well as to evaluate the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of the compounds in these extracts. The in vivo lethality test has proven effective as an initial assessment of cytotoxic and antitumor agents (Ramachandran et al., 2011). The compounds verified via GC-MS were also assessed for their ability to reach the brain and be absorbed in the gastrointestinal tract using the SWISSADME tool. This model predicts two crucial properties of a compound: its ability to permeate the blood-brain barrier (BBB), indicating whether it can reach the brain, and its gastrointestinal (GI) absorption, determining if it can be effectively absorbed in the GI tract, which is vital for oral bioavailability. The outcomes of this current study would provide fundamental insights into the most prospective plant extracts, serving as a foundation for developing novel therapeutic tools of significant importance. MATERIALS AND METHODS Study area The plant part was collected from the Rupandehi district of Nepal, which was identified as Kalanchoe pinnata at the Department of Botany, Amrit Science Campus, Thamel, Kathmandu, 44600 (Supplementary Table 1). Sample preparation About 4 kg of the plant's leaves were collected, cleaned, and air-dried for a few days. They were then shade-dried, ground into a fine powder (almost 400 g), and stored at a low temperature for later use. Extraction process The ultrasonic extraction process began with 400 g of powdered plant material in a sterile, dry beaker. Hexane was added, stirred, and the beaker was immersed in an ultrasonic cleaner tub (30×15×20 cm) filled one-third with distilled water. After extraction, the contents were decanted, cooled, and filtered. The filtrate was then concentrated using a rota evaporator, resulting in an extract that was measured, dried, and stored in airtight vials. This process was repeated three times using various solvents (dichloromethane, chloroform, ethyl acetate, acetone, methanol, and water), with hexane added to the filtered residue before sonication. Gas chromatography-mass spectroscopy Before undergoing GC-MS analysis, the concentrated extract obtained from hexane and methanol via the rota- evaporator was dissolved in chloroform. GC-MS was utilized to separate and analyze the multicomponent mixtures. Phytochemical screening The phytochemical analysis of various extracts was done based on the protocol by Banu and Cathrine in 2015 (Banu & Cathrine, 2015). Total phenolic content The overall phenolic content of the plant extract was determined using a colorimetric technique called Folin- Ciocalteu, which relies on an oxidation-reduction process. The gallic acid concentration was used as a benchmark. Milligrams of gallic acid equivalent per gram of dry extract (mg GAE/g of dry extract) were used to represent the sample's total phenolic content based on the gallic acid calibration curve. Initially, a stock solution was prepared with an extract concentration of 1000 μg/mL (ppm) by dissolving 1 milligram of extract in 1 mL of methanol. Subsequently, different concentrations of the extracts were prepared by serially diluting the stock solution, and their respective absorbance values were recorded (Balasundram et al., 2006). The following Formula 1 was used to determine the sample's total phenolic content, which was represented as milligrams of gallic acid equivalent per gram. 𝐶 = 𝑐𝑉/ 𝑚 (1) Where C is the total content of the phenolic compounds (mg/g) in gallic acid equivalent, c is the concentration of the gallic acid established from the calibration curve (μg/mL), V is the volume of the extract (mL), and m is the weight of the plant extract (μg). Total flavonoid content To quantify the total flavonoid content of the plant extract, an aluminum chloride colorimetric assay was performed (Chandra et al., 2014). Quercetin was used as the reference. Firstly, a stock solution, was prepared with an extract concentration of 1000 μg/mL (ppm) by dissolving 1 milligram of the extract in 1 mL of methanol. Following this, the stock solution was subjected to repeated dilution to produce a range of extract concentrations. The absorbance values of these diluted solutions were recorded. Bhusal et al. – Exploring the Therapeutic Potential of Kalanchoe Pinnata: … 619 Formula 2 was used to determine the sample's total flavonoid content, represented as milligrams of quercetin equivalents (QE) per gram of extract. 𝐶 = 𝑐𝑉/𝑚 (2) Where C is the total flavonoid content (mg/g) in Quercetin equivalent (QE), c is the concentration of quercetin established from the calibration curve (μg/mL), V is the Volume of the extract (mL), and m is the weight of the plant extract (μg). DPPH free radical scavenging activity The plant extracts were assessed for their ability to scavenge radicals using the spectrophotometric DPPH method (Sanna et al., 2012). The capacity to neutralize the DPPH radical was determined by observing the decrease in absorbance and calculated using the following Formula 3. 𝑅𝑎𝑑𝑖𝑐𝑎𝑙 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 (%) = [(𝐴0 − 𝐴𝑆)/𝐴0] × 100 (3) Where A0 is the absorbance of the control (DPPH solution + methanol) and AS is the absorbance of the test sample. The amount of an adequate sample needed to neutralize 50% of DPPH free radicals is known as the IC50 (50% inhibitory concentration) value. By graphing the extract concentration vs. the corresponding scavenging action, the inhibition curve and IC50 values were generated. 10 mg of each extract (chloroform, ethyl acetate, and methanol) were dissolved in 10 mL of methanol to create a stock solution of 1 mg/mL. This stock solution was then successively diluted to concentrations of 1000 μg/mL, 500 μg/mL, 250 μg/mL, and 125 μg/mL. For each concentration, 50 μL of ascorbic acid was mixed with 150 μL of methanolic DPPH solution in a 96-well plate, totaling 200 μL. After incubation in the dark for 20 minutes, the absorbance at 520 nm was measured using a spectrophotometer, with methanol and DPPH as blanks. Antimicrobial activity Microbial growth inhibition was evaluated using the paper disc diffusion method, and the outcome was quantified as a zone of inhibition (ZOI) value. In this assay, one Gram-positive bacterium (Bacillus subtilis: ATCC 6051), one Gram-negative bacterium (Escherichia coli: ATCC 8739), and a fungus (Candida albicans: ATCC 2091) were utilized. The efficacy of various K. pinnata leaf extracts in inhibiting microbial growth at a constant concentration (5 µg/mL) was evaluated. Screening and evaluation of antimicrobial activity Initially, 100 µL of culture broth from each strain was spread onto Mueller Hinton agar plates and incubated at 37 °C for 15 minutes. After incubation, the plates were left overnight at the same temperature. The antibacterial effects of the samples on B. subtilis, E. coli, and C. albicans strains were evaluated the next day. Kanamycin was the positive control, while DMSO was the negative control. The ZOI produced by each sample was measured using a scale. Alpha-amylase inhibition assay The α-amylase inhibitory potential of plant extracts was assessed using the 3,5-dinitrosalicylic acid (DNSA) technique. K. pinnata leaf extract was diluted with 10% DMSO to create different concentrations. These mixtures were further combined with buffer and NaCl at pH 6.9. The extract and α-amylase solution were mixed in a 200 μL volume and incubated at 30 °C for 10 minutes. The Starch solution was added and left for 3 minutes before stopping the process with the DNSA reagent. After heating in a water bath, the sample was diluted with distilled water and absorbance at 540 nm was measured using a UV spectrophotometer. The α-amylase inhibitory activity (%) was calculated using Formula 4. % 𝛼 − 𝑎𝑚𝑦𝑙𝑎𝑠𝑒 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝐴𝑏𝑠100%𝐶𝑜𝑛𝑡𝑟𝑜𝑙−𝐴𝑏𝑠𝑆𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑠100%𝐶𝑜𝑛𝑡𝑟𝑜𝑙 × 100 (4) Extract concentration was plotted against the percentage of α-amylase inhibition, and IC50 values were obtained from the resulting graph. Brine shrimp lethality assay (BSLA) The study employed a brine shrimp lethality bioassay to investigate the cytotoxicity of the plant extracts. Understanding the toxic effects of plant extracts is essential for safe treatment. The brine shrimp lethality test, utilizing Artemia salina, is a cost-effective and versatile method widely used to screen diverse chemical compounds for bioactivity (Waghulde et al., 2020). Before beginning the process, artificial seawater was created by combining the components in the specified amounts as detailed in Supplementary Table 2. Brine shrimp eggs, approximately 50 mg in weight, were dispersed over simulated seawater collected in a beaker, covered with aluminum foil, with a few small pores created to facilitate heat and light conductivity. The beaker was left at room temperature for 48 hours, exposed to radiation from a 60-watt bulb. A stock solution of 2 mg extract was prepared by adding 2 mL of DMSO (dimethyl sulfoxide), resulting in a concentration of 1000 ppm (mg/mL). 620 Biology, Medicine, & Natural Product Chemistry 13 (2), 2024: 617-625 Toxicities of compounds were tested at 1, 0.5, 0.125, and 0.0625 ppm in sea-water solutions with 2 mL DMSO. In each test, ten nauplii were used, and survivors were counted after 24 hours. Three replications were performed for each concentration. A blank control was conducted using DMSO. The lethal concentration for 50% mortality (LC50) after 24 hours of exposure, was determined using the probit method to measure the toxicity of the extract or fractions. LC50 values greater than 1000 ppm for plant extracts were considered inactive. The mortality percentage was then computed using the following Formula 5. % 𝑀𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 = 𝑁𝑜. of dead shrimps Total No. of shrimps × 100 (5) ADMET Properties In addition to conducting laboratory assays, the BOILED-Egg model was used for compounds verified through GC-MS analysis to determine their gastrointestinal absorption and blood-brain barrier (BBB) penetration capabilities (Dong et al., 2018). The canonical SMILES retrieved from PubChem (https://pubchem.ncbi.nlm.nih.gov/) for each compound were uploaded to the web server, and their properties were analyzed. Moreover, ADMET properties (absorption, distribution, metabolism, excretion, toxicity) were assessed using ADMET lab 2.0, and the toxicity class for each compound was identified with Protox 3.0 (https://tox.charite.de/protox3/). RESULTS AND DISCUSSION Gram yield The ultrasonication technique was utilized to extract compounds from the K. pinnata plant. The resulting extracts using hexane, dichloromethane, chloroform, ethyl acetate, acetone, methanol, and water showed varying yields: 7.31 g, 9.08 g, 2.21 g, 0.21 g, 1.04 g, 10.34 g, and 14.50 g, respectively (Supplementary Table 3). Qualitative analysis of phytochemicals The extracts of K. pinnata were analyzed for their phytochemical contents, and the presence of these compounds was confirmed through observable color changes. Among the extracts, varying compositions of volatile oils, alkaloids, carbohydrates, phenolic compounds, tannins, flavonoids, terpenoids, quinones, reducing sugar, and saponins were identified (Supplementary Table 4). The results of the phytochemical screening revealed that the polar extracts predominantly contained the highest concentrations of phytochemicals. Almost all solvent extracts tested positive for flavonoids and terpenoids. The absence of alkaloids in the extracts might be attributed to the method of extraction, which involved the use of an ultrasound sonicator followed by concentration using a rota-evaporator. This process could potentially lead to the decomposition of alkaloids due to heat and molecular vibration. The literature often presents slightly different findings compared to the results obtained. Discrepancies can arise due to several factors, including differences in plant altitude, varying environmental conditions, the specific extraction method employed, the timing of sample collection, differences in laboratory setups, and variations in chemical grades used during analysis. Consequently, the outcomes of phytochemical screenings for a particular sample may vary from one study to another, even when targeting the same constituents. GC-MS spectra analysis Hexane and methanol extract of K. pinnata were used for the GC-MS analysis. The analysis revealed seven primary components in the composition of the K. pinnata hexane extract (Table 1) (Supplementary Fig. 1 to Supplementary Fig. 8) and 20 components in the methanol extract (Table 2) (Supplementary Figure 9 to Supplementary Figure 29). The compounds along with their PubChem CID and canonical SMILES are presented in Supplementary Table 5. Table 1. Components present in the hexane extract according to GC-MS analysis. S.N. Name of compound Retention time Molecular formula Area (%) 1. 2H-1,2,3,4-Tetrazole-2-ethanol, .alpha.-(chloromethyl)-5-phenyl- 8.228 C10H11ClN4O 11.13 2. N-benzyl-2-(5-phenyl-2H-tetrazol-2-yl)acetamide 8.506 C16H15N5O 3.90 3. 2H-tetrazole-2-ethanole, 5-phenyl- 13.942 C9H10N4O 27.44 4. 1,4-Methanocycloocta[d]pyridazine, 1,4,4a,5,6,9,10,10a-octahydro-11,11- dimethyl-, (1.alpha.,4.alpha.,4a.alpha.,10a.alpha.)- 15.677 C13H20N2 11.98 5. Pentadecafluorooctanoicacid, dodec-2-en-1-yl ester 24.668 C20H23F15O2 29.36 6. Octanoic acid, 4-methyl-,ethyl ester,(.+/-.)- 26.96 C11H22O2 10.99 7. Dodecane, 2-methyl- 28.00 C13H28 5.19 Bhusal et al. – Exploring the Therapeutic Potential of Kalanchoe Pinnata: … 621 Table 2. Components present in the methanol extract according to GC-MS analysis. S.N. Name of compound Retention time Molecular formula Area (%) 1. 2H-Tetrazole-2-ethanole, 5-phenyl- 9.542 C9H10N4O 19.61 2. Carbon dioxide 6.425 CO2 9.51 3. 1,4-Methanocycloocta[d]pyridazine, 1,4,4a,5,6,9,10,10a-octahydro- 11,11-dimethyl-, (1.alpha.,4.alpha.,4a.alpha.,10a.alpha.)- 16.229 C13H20N2 6.29 4. 1-bromo-2-phenyl-cyclopropanphosphonic acid, diethyl ester 15.281 C13H18BrO3P 0.70 5. 4-Diethylaminophenyl isothiocyanate 18.255 C11H14N2S 0.82 6. Pentadecafluorooctanoic acid, undecyl ester 19.710 C19H23F15O2 0.79 7. Oxirane, octyl- 21.876 C10H20O 0.76 8. Methyl 6-methyloctanoate 22.244 C10H20O2 0.89 9. alpha-Methyl-alpha-[4-methylpentyl]oxiranmethanol 22.858 C10H20O2 0.74 10. Bacteriochlorophyll-c-stearyl 25.126 C52H72MgN4O4 1.71 11. Levomenthol 24.284 C10H20O 1.68 12. 2-Isopropenyl-5-methyl-6-hepten-1-ol 25.028 C11H20O 0.67 13. 13-Borabicyclo[7.3.0]tridecane, 13-butoxy-, (Z)-or (E)- 25.439 C16H31BO 0.61 14. 8-Methylnonanoic acid, methyl ester 26.555 C11H22O2 22.38 15. Decanoic acid, silver (1+) salt 26.285 C10H19AgO2 1.07 16. 1,2-Oxathiane, 6-dodecyl-,2,2-dioxide 27.204 C16H32O3S 0.92 17. Methyl-9,10-octadecadienoate 28.517 C19H34O2 14.50 18. Cyclododecyne 28.639 C12H20 9.65 19. 3,7-Dimethyl-6-nonen-1-ol 28.799 C11H22O 2.46 20. Decanoic acid, methyl ester 28.930 C11H22O2 3.73 Estimation of total phenolic and flavonoid content The results indicated that the methanol extract of K. pinnata contains 182.87 mg GAE/g, while the ethyl acetate extract contains 485.17 mg GAE/g of phenolic content. These findings imply that phenolic compounds generally show higher solubility in polar organic solvents compared to non-polar ones. Additionally, ethyl acetate displayed a markedly higher total flavonoid concentration of 40.25 mg QE/g, in contrast to methanol, which showed a concentration of 19.05 mg QE/g (Table 3). Table 3. Total phenolic and flavonoid content in ethyl acetate and methanolic extract of K. pinnata leaves. Extract Ethyl acetate Methanol Total phenolic content (mg GAE /g extract) 485.17 182.87 Total flavonoid content (mg QE/g extract) 40.25 19.05 Antioxidant activity The chloroform extract of K. pinnata demonstrated an IC50 value of 2219.41 µg/mL, indicating relatively low antioxidant activity. In contrast, the ethyl acetate extract showed a much lower IC50 value of 365.22 µg/mL (Figure 1), suggesting stronger antioxidant properties. The methanol extract exhibited the highest antioxidant activity with an IC50 value of 293.53 µg/mL at 520 nm absorbance. These IC50 values reflect the varying effectiveness of the extracts in scavenging free radicals, with lower values indicating greater antioxidant potency. 0 500 1000 1500 2000 2500 Chloroform Ethyl acetate Methanol IC 5 0 µ g /m L Extracts Figure 1. IC50 values of different extracts (chloroform, ethyl acetate, and methanol) of K. pinnata. Antimicrobial activity The antimicrobial activity of various extracts was measured by the diameter of the inhibition zone (ZOI) in centimeters, compared to the negative control (DMSO). For Bacillus subtilis, the hexane, dichloromethane (DCM), chloroform, and acetone extracts showed inhibition zones ranging from 0.70 to 0.85 cm, indicating moderate antimicrobial activity. The ethyl acetate, methanol, and aqueous extracts displayed smaller inhibition zones of 0.50 cm. In the case of Escherichia coli, both the hexane and methanol extracts had the largest inhibition zones at 0.80 cm, while the other extracts ranged between 0.60 and 0.70 cm. For Candida albicans, the hexane, DCM, and acetone extracts exhibited the largest inhibition zones of 0.75 cm, while the ethyl acetate and aqueous extracts had smaller inhibition zones of 0.60 and 0.50 cm, respectively. 622 Biology, Medicine, & Natural Product Chemistry 13 (2), 2024: 617-625 Overall, the extracts varied in their antimicrobial efficacy across different microbes. The effectiveness of extracts varied with the type of microbe. For example, hexane and acetone extracts showed higher inhibition against Candida albicans compared to other extracts, suggesting that certain extracts might be more effective against specific microbes. Table 4. Antimicrobial activity is shown by the different extracts in diameter (cm). Microbes Negative control DMSO Hexane extract DCM extract Chloroform extract Ethyl acetate extract Acetone extract Methanol extract Aqueous extract Bacillus subtilis (ATCC 6051) 0 0.85 0.80 0.85 0.7 0.85 0.50 0.50 Escherichia coli (ATCC 8739) 0 0.65 0.80 0.70 0.65 0.60 0.80 0.70 Candida albicans (ATCC 2091) 0 0.70 0.75 0.60 0.60 0.75 0.70 0.50 Alpha-amylase inhibition assay In the alpha-amylase inhibition assay, K. pinnata acetone extract displayed an IC50 of 285.71 µg/mL, while ethyl acetate extract showed 504.21 µg/mL, and methanol extract exhibited a value of 195.39 µg/mL (Figure 2). Comparatively, among these three extracts, the methanol extract of K. pinnata showed higher α-amylase inhibition activity. 0 100 200 300 400 500 600 Acetone Ethyl acetate Methanol IC 5 0 va lu e s Extracts Figure 2. IC50 values of different extracts for α-amylase inhibition. Cytotoxicity activity After the freshly hatched live nauplii were exposed to concentrations of 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, and 0.0625 mg/mL, respectively, the LC50 value of chloroform, acetone, and methanol extracts was computed (Table 5). It was found that the concentration of the extract directly correlated with the level of lethality (Supplementary Table 6 to Supplementary Table 8). The highest and lowest numbers of s brine shrimp larvae perished at doses of 1 mg/mL and 0.0625 mg/mL, respectively. Table 5. Lethality concentration of 3 different extracts of K. Pinnata. Extracts LC50 (mg/mL) Chloroform 0.18 Acetone 0.17 Methanol 0.29 Among the three extracts, the chloroform extract exhibited a high mortality rate of 96.67%, while the methanol extract showed the lowest mortality rate of 56.66% at the extract concentration of 0.5 mg/mL. The LC50 values for the chloroform, acetone, and methanol extracts of K. pinnata were determined to be 0.181 Bhusal et al. – Exploring the Therapeutic Potential of Kalanchoe Pinnata: … 623 mg/mL, 0.175 mg/mL, and 0.29 mg/mL, respectively (Table 5). As a result, extracts were observed to be pharmacologically significant and harmful to Artemia salina (brine shrimp) larvae. The cytotoxic properties of these extracts may be attributed to the presence of alkaloids, tannins, and flavonoids (Waghulde et al., 2020). The significant lethality observed in various plant extracts towards brine shrimp implies the existence of potent cytotoxic compounds, highlighting the need for additional investigation. ADMET Analysis According to the BOILED-Egg model, the majority of molecules were concentrated within the yellow and white regions, indicating they possess balanced WLOGP and TPSA values. Out of the 27 compounds, 3 were found to be outside of the range as seen in the BOILED-Egg model. Molecule 9 was located at the lower end of the WLOGP scale (below -1), indicating it has low lipophilicity. As a PGP+ substrate, it might be actively transported out of cells, which could impact its bioavailability and penetration. Therefore, its low lipophilicity and the possibility of being actively transported out of cells make it an outlier compared to the other molecules. Such status implies that it may have limited absorption or distribution characteristics that are favorable for central nervous system (CNS)-targeted therapies. The other two outliers, Molecule 1 and Molecule 2, were positioned towards the higher end of the TPSA scale (around 100), indicating a larger polar surface area (Figure 3). This correlates with lower membrane permeability, which can affect their ability to be absorbed in the intestine and penetrate the BBB. Their high TPSA values make them outliers, as they fall outside the optimal range for HIA and BBB penetration. All the compounds were found to fall into Class 4 toxicity, indicating they have low toxicity but can be detrimental if given in high doses according to the ProTox3.0 server. This classification suggests that these compounds are relatively safe and pose a low risk of causing significant harm at typical exposure levels. Figure 3. BOILED-Egg model for the identified 27 compounds. Similarly, the ADMET properties of molecules extracted from hexane and methanol were analyzed. All molecules generally adhered to Lipinski's Rule of Five, indicating good drug-like properties. The majority of compounds exhibited excellent Caco-2 permeability and were classified as CYP3A4 substrates and inhibitors, suggesting effective absorption and metabolism. However, there was variability in their carcinogenic potential, ranging from non-carcinogenic to carcinogenic (Supplementary Table 9). Clearance rates were predominantly excellent, though a few molecules (6 out of 27) showed poor clearance, which could impact their overall efficacy and safety profiles. Molecule 5 was rejected by Lipinski’s rule, indicating poor drug-like qualities, although it had excellent permeability and clearance. Molecule 9, while passing Lipinski’s rule and showing excellent permeability, suffered from poor clearance, which potentially impacted its effectiveness. Molecule 17 was rejected by Lipinski’s rule and had both poor permeability and clearance, suggesting significant issues with absorption and excretion. These factors highlighted potential challenges for these molecules in terms of drug development and effectiveness. 624 Biology, Medicine, & Natural Product Chemistry 13 (2), 2024: 617-625 CONCLUSIONS This study comprehensively evaluates the biological activities and phytochemical composition of Kalanchoe pinnata leaf extracts, highlighting their potential as therapeutic agents. The phytochemical analysis confirmed the presence of a diverse array of bioactive compounds, including flavonoids, phenolics, and terpenoids, across various extracts. Notably, the methanol and ethyl acetate extracts exhibited substantial antioxidant activity, with IC50 values indicating significant radical scavenging potential. The ethyl acetate extract, in particular, demonstrated the highest phenolic and flavonoid contents, correlating with its potent antioxidant effects. The alpha-amylase inhibition assay revealed that the methanol extract had superior α- amylase inhibitory activity, suggesting its potential for diabetes management. The brine shrimp lethality assay underscored significant toxic effects in the chloroform and acetone extracts, highlighting the need for further investigation into their mechanisms and safety. ADMET analysis showed that K. pinnata extracts generally exhibited favorable absorption and distribution profiles, though toxicity predictions indicated potential risks depending on dosage and exposure. The results emphasized the need for further research to balance therapeutic benefits with safety. Overall, K. pinnata demonstrated considerable promise for antioxidant, antimicrobial, and antidiabetic applications, with future studies needed to refine safety and efficacy while considering ADMET factors. Acknowledgements: The authors express their gratitude to the Department of Plant Resources, Kathmandu, Nepal for conducting the GC-MS experiments. Authors’ Contributions: Conceptualization: Ram Lal Swagat Shrestha; Methodology: Dakshina Bhusal, Ashika Tamang, Timila Shrestha, Samjhana Bharati, and Binita Maharjan; Discussion of results: Sujan Dhital and Nirmal Parajuli; Writing Original Draft: Dakshina Bhusal, Manila Poudel; Review and Editing: Jhashanath Adhikari Subin, Bishnu Prasad Marasini, and Ram Lal Swagat Shrestha. All authors read and approved the final version of the manuscript. Competing Interests: The authors declare that there are no competing interests. Funding: The authors declare that this study did not receive any funding. REFERENCES Arroyave, F., Montaño, D., & Lizcano, F. (2020). Diabetes mellitus is a chronic disease that can benefit from therapy with induced pluripotent stem cells. International Journal of Molecular Sciences, 21(22), 1–28. https://doi.org/10.3390/ijms21228685 Balasundram, N., Sundram, K., & Samman, S. (2006). 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