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INTRODUCTION
The use of medicinal plants for the treatment of various ailments has been deeply embedded in Indian culture since ancient times. For last many centuries, the well-established treatment systems like Ayurveda, Siddha and Unani are being practiced in India and since then these systems are playing important role in public healthcare. In these systems of treatment different parts of plants like roots, leaves, stems, seeds, flowers etc. are used to different types of healthcare issues.
At present, several Ayurvedic medicines derived and prepared from different plant formulations gained acceptance in modern medicine and reached to global market places (Patwardhan et al., 2005). Therefore, plants serve as vital raw materials in the development of pharmaceutical drugs. Though the synthetic drugs are effective in managing many diseases, they are often expensive and also comes with different side effects.
As of now, it is estimated that nearly 70,000 plant species have been used for medicinal purposes worldwide. As these plants contains, complex phytochemical constituents like alkaloids, flavonoids, tannins, terpenoids, and glycosides are the main reason behind such acceptance and use.
In India too more than 2,500 plant species have been recognized for its medicinal importance. Similarly, Sri Lanka and Nepal too have confirmed 1,400 and 700 species, respectively. Hence, these plants have plenty of bioactive compounds, however, the scientific research of many of which remain underexplored. Thus, the interest in natural remedies and alternative therapies is increasing the integration of traditional knowledge with modern pharmacological approaches opens up a promising avenue for drug discovery and healthcare discoveries. Among these, the genus Euphorbia is one of the largest and most pharmacologically potent, with over 2,000 species spread across various regions globally (Nabeelah et al., 2019). Euphorbia microphylla B. Heyne ex Roth, commonly known in some regions as Hayna, is a lesser-known but it is widely used species in traditional medicine. It belongs to the family Euphorbiaceae, characterized by the presence of milky latex, cyathium inflorescence, and a huge diversity of bioactive compounds. According to Althobaiti A.T., (2023) approximately 1,600 species of Euphorbia distributed globally. Euphorbia includes flavonoids, triterpenoids, alkanes, amino acids, and alkaloids, which possesses a range of pharmacological effects (Kamboj, 2000).
The Euphorbia microphylla is found primarily in dry and semi-dry regions, flourishing in poor soils and extreme climatic conditions. In traditional medicine system. it is commonly used for treating skin ailments, gastrointestinal issues, inflammation, and even tumours (Kgosiemang et al., 2025). Despite its regular use in folk remedies, scientific evaluation of its phytochemical composition, pharmacological efficacy, and toxicological safety is not explored on large scale.
Ethnobotanical and Traditional Uses
Traditionally, especially among indigenous and rural communities, Euphorbia microphylla (Hayna) has been used for different therapeutic applications like wound healing, Anti-inflammatory treatments, laxative and purgative effects, skin diseases, fever and treatment of gastrointestinal disorders.
Due to its irritant and caustic natures, the latex is used in diluted forms. As it contain repellent properties, in certain African and South Asian cultures, for and also in religious rituals. However, despite its popularity in folk medicine, scientific studies validating these uses are rare and thus underlines the need for pharmacological and toxicological assessments. The plant's latex, though toxic in high doses, is often diluted in small quantity and applied to treat warts, fungal infections, and even tumour in some folk traditions. On the other hand, in some cultures, the roots and leaves are used in decoctions or pastes which is often mixed with other herbs (Amtaghri et al., 2022),
Phytochemical Composition
The plant is rich in bioactive compounds like diterpenoids, flavonoids, alkaloids, tannins, steroids, saponins (Alabri et al., 2018).
Crude extract of Euphorbia microphylla identified several compounds with known anti-inflammatory and antimicrobial activities. These compounds are responsible for the plant’s antimicrobial, anti-inflammatory, antioxidant, and cytotoxic activities (Benjamaa, et al., 2024). Continued phytochemical screening is necessary to isolate and identify novel compounds with potential pharmaceutical value.
Pharmacological Activities
Preliminary investigations on pharmacological activities of Euphorbia suggest the various activities as anti-inflammatory by inhibiting of prostaglandin pathways (Das et al., 2022), antimicrobial i.e. Latex inhibits bacterial and fungal growth antioxidant because of flavonoids and polyphenols scavenge free radicals, and cytotoxicity because of some diterpenes exhibit selective cytotoxicity against cancer cells (Benjamaa et al., 2022).
These findings need to be continued with phytochemical screening necessary to isolate and identify novel compounds with potential pharmaceutical value. The study also need to validate through controlled in vitro and in vivo experiments to ascertain dosage safety, efficacy, and side effects (Figure 1).
MATERIALS AND METHODS
The collection of plant material, proximate analysis, extraction, yield calculation, pH determination, and phytochemical screening was conducted by following methods,
Collection of plant material
The plant leaves of Euphorbia microphylla were collected in accordance with local, national, and international guidelines and regulations for plant research the month of December 2024 from Sangli, MS and identified at Biocyte Research and Development Pvt. ltd, Sangali, Maharashtra. The species is not listed as endangered or protected, and no specific permits were required for its collection. Plant leaves were washed properly using distilled water, dried under shade and grinded to fine powder. The powder was sieved and a portion of the powder was used for proximate principle analysis and the other portion was subjected to solvent extraction.
Proximate principle analysis
The dried sample of E. microphylla was analysed for proximate principles like total Ash, Acid Insoluble Ash, Water-Soluble Extractive substances, Alcohol-Soluble Extractive substances, and pH (1% Solution). These parameters ensure the purity and stability of the drug, helping to detect adulteration and degradation (Park and Bell, 2004).
Extraction
Euphorbia microphylla leaves were collected, washed with distilled water, and dried in the shade at room temperature or in a hot air oven at 40-45ºC for 7-10 days. The dried leaves were crushed to a rough powder and 30-50 g of powder was weighed for extraction. The powdered sample was placed in a Soxhlet apparatus thimble, and 250-300 mL of 70-95% ethanol was added to the round-bottom flask. The Soxhlet extractor was assembled, connected with a condenser, and heated to maintain the solvent at its boiling point (~78ºC). Extraction was carried out for 6-8 hr or until the siphoning solvent became colourless, indicating complete extraction. After extraction, the solution was cooled, filtered through Whatman No. 1 filter paper to remove residues, and the filtrate was concentrated using a rotary evaporator under reduced pressure or evaporated over a water bath at 40-50ºC. The concentrated extract was dried completely in a desiccator and stored in an airtight amber glass container at 4ºC for further analysis. The yield of crude extract was typically 5-15% w/w of dried plant material.
Calculation of extractive yield
The weight of the dried crude ethanolic and hexanic extracts was recorded and the extractive yield was calculated as under Loss on Drying/ total ash/ Acid Insoluble Ash (Harborne, 1998).
(W1 is the weight of the empty crucible, W2 is the weight of the crucible plus sample before ashing/drying, and W3 is the weight of the crucible plus ash after incineration/drying.)
The Water-Soluble Extractive and Alcohol-Soluble Extractive (%) is then calculated using the formula:
pH determination
pH of a 1% w/v (weight/volume) aqueous solution of the sample, which helps assess its acidity or alkalinity by using pH meter.
Qualitative phytochemical screening
Qualitative tests were conducted on all extracts to identify the various phytoconstituents present. The tests performed and the reagents used as per the method given by Harborne A J (1998).
Quantitative phytochemical analysis
Phenols
To estimate the phenol content in a sample, the Folin-Ciocalteu method is used, which relies on the reduction of the Folin-Ciocalteu reagent by phenolic compounds to produce a blue complex measurable by spectrophotometry. The flavonoid content is calculated by plotting the absorbance values of the standards to generate a standard curve, from which the sample’s flavonoid concentration is determined (Madhu et al., 2016).
Flavonoid
To estimate the flavonoid content in a sample, the aluminum chloride colorimetric method is widely used, as it relies on the formation of a yellow complex between flavonoids and aluminum chloride that can be measured spectro-photometrically. The flavonoid content is calculated by plotting the absorbance values of the standards to generate a standard curve, from which the sample’s flavonoid concentration is determined (Isaac et al., 2024).
Statistical analysis
Results were expressed as mean±Standard Deviation (SD). The SD was calculated to assess the variability and precision of the experimental measurements.
In vitro anti-inflammatory activity
In vitro anti-inflammatory activity was conducted by three different methods i.e. protein denaturation inhibition assay (Dharmadeva et al., 2018) protease inhibition assay (Sohemat et al., 2023) and membrane stabilization assay (Chippada et al., 2011).
Statistical Analysis
Data were analyzed using one-way ANOVA and expressed as Mean±SD of triplicate determinations. IC₅₀ values were calculated by regression analysis, and p<0.05 was considered statistically significant.
RESULTS
Biochemical proximate principles in dried E. microphylla Organoleptic and Physicochemical Parameters
The evaluation of Euphorbia microphylla revealed typical organoleptic characteristics, with a greenish- brown colour, characteristic slightly pungent smell, coarse powder or dried plant material appearance, and a bitter, astringent taste. Physicochemical analysis showed acceptable parameters for herbal material, with a moisture content of 5.7%, total ash value of 5.4%, and acid insoluble ash of 0.5%, indicating low contamination with inorganic matter. In alcohol, the extractive values were significant followed by water. The pH value was observed as acidic. the findings confirm the identity, purity, and quality of the Euphorbia microphylla sample for further pharmacognostical or phytochemical investigations (Tables 1,2).
| Sl. No. | Sample | Parameter | |||||
|---|---|---|---|---|---|---|---|
| Moisture content / Loss on soiling % | Total Ash value % | Acid insoluble ash | Alcohol soluble extractive | Water soluble extractive | pH | ||
| 1 | Euphorbia microphylla | 5.7 | 5.4 | 0.5 | 15 | 7.5 | 5.9 |
| Sl. No. | Sample code | Parameter | |||
|---|---|---|---|---|---|
| Colour | Smell | Appearance | Taste | ||
| 1 | Euphorbia microphylla | Greenish- brown (typical of dried herb) | Characteristic, slightly pungent | Coarse powder / dried plant material (depending on form) | Bitter, astringent |
Organoleptic Characters
Physicochemical Parameters
Qualitative phytochemical screening
To identifying the bioactive compounds in plant and also to assess its medicinal importance, phytochemical analysis is necessary. Such identification forms the basis for further targeted research and important compound isolation. The solvent which is used in this process determines the effectiveness of the phytochemical extraction. On the other hand, the qualitative tests confirms the presence or absence of specific phytochemicals. Therefore, in the present study, Euphorbia microphylla leaves tested positive for alkaloids, carbohydrates, glycosides, and saponins, suggesting potential pharmacological significance. However, it tested negative for proteins, amino acids, reducing sugars, flavonoids, cardiac glycosides, phenolic compounds, and tannins. These findings provide valuable insights for future studies on the therapeutic applications of Euphorbia microphylla (Table 3).
| Sl. No. | Component | Test Performed | Confirmation Test |
|---|---|---|---|
| Carbohydrate | Molisch’s test Test for starch | +VE +VE | |
| Alkaloids | Wagner’s test Hager’s test | -VE ++VE | |
| Reducing sugars | Benedict’s test Fehling’s test | +VE -VE | |
| Glycosides | 10% NaOH test Aqueous NaOH test | -VE +VE | |
| Cardiac Glycosides | Baljet test Test for Cardenolides | -VE +VE | |
| Proteins | Biuret test Millons Test | -VE +VE | |
| Flavonoids | Lead acetate test Ferric chloride test | ++VE ++VE | |
| Phenolic compounds | Ferric chloride test Hot water test | ++VE ++VE | |
| Tannins | Braymer’s test 10% NaOH test | -VE -VE | |
| Saponins | Foam test Olive oil test | +VE +VE |
Quantitative phytochemical analysis
Phenolic compounds
During the study it was observed that the standard curve of gallic acid showed a linear increase in Optical Density (OD). Further, when concentration was increased, it confirms the reliability of the method for phenolic content estimation. The ethanolic extract of Euphorbia microphylla showed a mean OD of 0.93 at a concentration of 1000 µg/mL. Based on the standard curve, the total phenolic content of the extract was found to be 1.896 µg GA/g, equivalent to 0.1896 mg GA/g. The phenolic content is further expressed as mg GAE/g±SD, with a value of 0.1896 (Tables 4, 5 and Figure 2).
| Sl. No. | Gallic acid Concentration (µg/mL) | Gallic acid Concentration (mg/mL) | OD |
|---|---|---|---|
| 1 | 200 | 0.2 | 0.1 |
| 2 | 400 | 0.4 | 0.24 |
| 3 | 600 | 0.6 | 0.33 |
| 4 | 800 | 0.8 | 0.42 |
| 5 | 1000 | 0.1 | 0.52 |
| Sl. No . | Samples at Concentration (1000 µg/mL) | OD | Mean OD | Total phenolic contents (µg GA/g) | Total phen olic contents (mgGA/g) in±SD |
|---|---|---|---|---|---|
| 1 | Ethanolic Extract of Euphorbia microphylla | 0.97 0.93 0.91 | 0.93 | 1.896 | 0.1896 |
Flavonoid Content
The standard curve of quercetin demonstrated a clear linear increase in Optical Density (OD). While when the concentration of quercetin increased, it confirmed the method’s suitability for flavonoid content analysis. For the ethanolic extract of Euphorbia microphylla, the mean OD at a concentration of 1000 µg/mL was 0.16. Based on the standard curve, the flavonoid content in the extract was calculated to be 260 µg Quercetin Equivalent (QE)/g, which is equivalent to 0.260 mg QE/g. These results shows that Euphorbia microphylla is a prominent source of flavonoids (Tables 6, 7 and Figure 3).
| Sl. No. | Quercetin Concentration (mg/mL) | Quercetin Concentration (µg/mL) | OD |
|---|---|---|---|
| 1 | 0.2 | 200 | 0.08 |
| 2 | 0.4 | 400 | 0.23 |
| 3 | 0.6 | 600 | 0.35 |
| 4 | 0.8 | 800 | 0.45 |
| 5 | 0.1 | 1000 | 0.59 |
| Sl. No. | Samples at Concentration (1000 µg/mL) | Absorbance (OD) | Mean (OD) | Flavonoid content (µg quercetin equivalent /g dry material) | Flavonoidcontent (mg quercetin equivalent /g dry material) |
|---|---|---|---|---|---|
| 1 | Ethanolic Extract of Euphorbia microphylla | 0.17 0.15 0.18 | 0.16 | 260 µg QE/g dry | 0.260 mg QE/g |
In vitro anti-inflammatory activity
Protein Denaturation Inhibition Assay
The ethanolic extract of Euphorbia microphylla showed significant protein denaturation inhibition and when the concentration increased, it leads to a higher percentage of inhibition. At a concentration of 200 µg/mL, the extract inhibited 43.34% of protein denaturation, and this effect increased gradually to 76.45% at 1000 µg/mL. The IC50 value for the ethanolic extract was found to be 365.05 µg/mL, which indicates its moderate inhibitory activity compared to the standard diclofenac sodium, i.e., IC50 of 77.14 µg/mL. These results suggest that Euphorbia microphylla possesses potential for protein denaturation inhibition (Table 8 and Figures 4,5).
| Sl. No. | Sample | Conc. | O.D. (Triplicate) | Mean±SD | % inhibition | IC50 | ||
|---|---|---|---|---|---|---|---|---|
| I | II | III | ||||||
| 1 | Control | 0.98 | 0.99 | 0.96 | 0.97±0.015 | |||
| 2 | Std Diclofenac Sodium | 200 | 0.49 | 0.52 | 0.48 | 0.49±0.015 | 49.14 | 77.14 |
| 400 | 0.29 | 0.28 | 0.27 | 0.28±0.01 | 71.33 | |||
| 600 | 0.15 | 0.14 | 0.12 | 0.13±0.015 | 86 | |||
| 800 | 0.11 | 0.09 | 0.08 | 0.09±0.015 | 90.44 | |||
| 1000 | 0.08 | 0.05 | 0.07 | 0.06±0.015 | 93.17 | |||
| 3 | Ethanolic Extract of Euphorbia microphylla | 200 | 0.57 | 0.55 | 0.54 | 0.55±0.01 | 43.34 | 365.05 |
| 400 | 0.52 | 0.49 | 0.45 | 0.48±0.01 | 50.17 | |||
| 600 | 0.42 | 0.39 | 0.35 | 0.38±0.01 | 60.4 | |||
| 800 | 0.32 | 0.29 | 0.25 | 0.28±0.015 | 70.64 | |||
| 1000 | 0.24 | 0.22 | 0.23 | 0.23±0.015 | 76.45 |
Proteinase Inhibitory Activities
The ethanolic extract of Euphorbia microphylla demonstrated notable proteinase inhibitory activity and concentration-dependent increased inhibition. At 200 µg/mL, the extract exhibited 34.59% inhibition, which increased to 75.75% at 1000 µg/mL. The IC50 value of the extract was calculated to be 438.87 µg/mL, that indicated moderate inhibitory potency compared to the standard drug ibuprofen i.e. IC50 of 171.41 µg/mL. These findings suggest that Euphorbia microphylla holds potential anti-inflammatory properties through proteinase inhibition. Although its activity is lower than that of standard anti-inflammatory drugs (Table 9 and Figures 6-8).
| Sl. No. | Sample | Conc. | O.D. (Triplicate) | Mean±SD | % inhibition | IC50 | ||
|---|---|---|---|---|---|---|---|---|
| I | II | III | ||||||
| 1 | Control | 0.896 | 0.889 | 0.892 | 0.892±0.003 | |||
| 2 | Std Ibuprofen | 200 | 0.456 | 0.455 | 0.458 | 0.456±0.001 | 48.86 | 171.41 |
| 400 | 0.345 | 0.348 | 0.348 | 0.347±0.001 | 61.11 | |||
| 600 | 0.225 | 0.228 | 0.219 | 0.224±0.004 | 74.89 | |||
| 800 | 0.198 | 0.199 | 0.197 | 0.198±0.001 | 77.81 | |||
| 1000 | 0.112 | 0.116 | 0.118 | 0.115±0.003 | 87.07 | |||
| 3 | Extract of Euphorbia Microphylla | 200 | 0.589 | 0.584 | 0.578 | 0.583±0.01 | 34.59 | 438.87 |
| 400 | 0.468 | 0.467 | 0.456 | 0.463±0.01 | 48.03 | |||
| 600 | 0.342 | 0.335 | 0.332 | 0.336±0.01 | 62.3 | |||
| 800 | 0.256 | 0.242 | 0.241 | 0.246±0.02 | 72.39 | |||
| 1000 | 0.213 | 0.223 | 0.213 | 0.216±0.01 | 75.75 |
Membrane Stabilization Assay
The ethanolic extract of Euphorbia microphylla demonstrated moderate membrane stabilization activity in a concentration-dependent manner. At 200 µg/mL, the extract showed 33.91% inhibition of hemolysis, which increased to 65.38% at 1000 µg/mL. The IC50 value of the extract was found to be 653.18 µg/mL, indicating comparatively lower potency than the standard drug, which exhibited an IC50 of 365.39 µg/mL. Although the membrane stabilizing effect of Euphorbia microphylla was weaker than that of the standard, the results suggest the extract still possesses potential anti-inflammatory properties by protecting red blood cell membranes against lysis under stress condition (Table 10 and Figures 9, 10).
| Sl. No. | Sample | Conc. | O.D. (Triplicate) | Mean±SD | % inhibition | IC50 | ||
|---|---|---|---|---|---|---|---|---|
| I | II | III | ||||||
| 1 | Control | 0.98 | 0.92 | 0.96 | 0.95±0.03 | |||
| 2 | Std | 200 | 0.56 | 0.58 | 0.59 | 0.57±0.015 | 39.51 | 365.39 |
| 400 | 0.48 | 0.44 | 0.42 | 0.44±0.03 | 53.14 | |||
| 600 | 0.37 | 0.32 | 0.34 | 0.34±0.02 | 63.98 | |||
| 800 | 0.29 | 0.27 | 0.25 | 0.27±0.02 | 71.67 | |||
| 1000 | 0.22 | 0.19 | 0.18 | 0.19±0.02 | 79.37 | |||
| 3 | Extract of Euphorbia microphylla | 200 | 0.62 | 0.63 | 0.64 | 0.6357±0.02 | 33.91 | 653.18 |
| 400 | 0.58 | 0.59 | 0.55 | 0.5757±0.01 | 39.86 | |||
| 600 | 0.53 | 0.51 | 0.49 | 0.5157±0.02 | 46.5 | |||
| 800 | 0.42 | 0.45 | 0.44 | 0.4357±0.03 | 54.19 | |||
| 1000 | 0.32 | 0.3 | 0.37 | 0.3357±0.01 | 65.38 |
Ethical Statement
This study did not involve human participants.
DISCUSSION
The qualitative phytochemical screening of the plant extract E. microphylla revealed the presence variety of secondary metabolites i.e., carbohydrates, reducing and non-reducing sugars, proteins, flavonoids, glycosides, saponins, steroids, tannins, phenolic compounds, alkaloids and triterpenoids. These earlier reports suggests that medicinal plants are biological factories for multiple classes of phytochemicals, which have potent biological activities (Peiris et al., 2023). In the screening of phytochemicals, flavonoids, phenolic compounds and glycosides were found prominent. Flavonoids play key roles as antioxidants and antimicrobial agents. Plant-derived flavonoids are effective in membrane disruption of microbial cells and interfering in oxidative pathways. The studies on detection of glycosides and saponins further supports the potential for pharmacological activity (Alhaithloul, 2023).
During the qualitative screening it is important to focus on reliability of qualitative phytochemical screening sample preparation, extraction solvent, test conditions and storage of material (Peiris et al., 2023). In the present study, standard chemical tests i.e. Molisch, Benedict’s, Seliwanoff’s, Biuret, Xanthoproteic, foam test, Salkowski’s, Keller-Killiani’s, Wagner’s etc. were used to screen the phytoconstituents. These tests gave rapid preliminary information. Further, chromatographic or spectrometric analyses (HPLC, GC-MS, NMR) is required to characterise and quantify each constituent (Kumar et al., 2023).
The presence of active secondary metabolites suggests that the plant extract has potential to exhibit multi-faceted pharmacological effects E.g. antioxidant, antimicrobial, anti-inflammatory etc. A recent Ethiopian study found that flavonoids and phenols were dominant in medicinal plant extracts demonstrating antimicrobial efficacy (Dubale et al., 2023).
While studying three types of anti-inflammatory assays, i.e. protein denaturation inhibition assay, protease inhibition assay and membrane stabilisation assay following points are discussed.
The protein denaturation inhibition assay evaluates the activity of plant extracts to prevent heat-induced unfolding of proteins like Bovine Serum Albumin (BSA) or egg albumin which is a key mechanism in inflammation where denatured proteins act as autoantigens in conditions resembling with rheumatoid arthritis. Ethanolic extracts from plants i.e. Euphorbia microphylla, viz. rich in phenolics and flavonoids, likely inhibit this process in a concentration-dependent manner, as compared to standards like diclofenac sodium. Similar studies were conducted on Ficus racemosa bark where inhibition increased with concentration up to 1000 μg/mL (Dharmadeva et al., 2018), confirming the method's reliability for screening anti-inflammatory potential in leaf extracts.
Protease inhibition assay measures suppression of enzymes in degradation of proteins like trypsin during inflammation. It also reduces the tissue damage and migration of immune cell. Hence, based on this principle, the study was performed on plant extracts that showed significant inhibition with IC50 values close with diclofenac sodium (93 μg/mL). This confirms that polyphenolics acts as natural protease inhibitors. During study of ethanolic extract of Euphorbia microphylla, this supports anti-inflammatory efficacy (Sohemat et al., 2023), in which aqueous extracts demonstrated dose-dependent protection. In membrane stabilization assay protection of Human Red Blood Cell (HRBC) membranes from hypotonic lysis, mimicking lysosomal membrane stability in inflamed tissues is assessed. Studiy conducted on extracts of Centella asiatica showed up to 94.97% stabilization at 2000 μg/mL, due to flavonoids preventing lipid peroxidation (Chippada et al., 2011). In Euphorbia microphylla, high phenolic content shows similar results validating for ethanolic leaf extracts.
CONCLUSION
The detailed evaluation of Euphorbia microphylla leaves confirms it’s pharmacological potential. Similar to a typical herbal material, Euphorbia too shows the organoleptic characteristics i.e., greenish-brown colour, pungent smell, and bitter, astringent taste. Physicochemical parameters such as low moisture content (5.7%), total ash (5.4%), and acid-insoluble ash (0.5%) indicate the material's quality and suitability for medicinal use.
On the other hand, phytochemical analysis confirms the presence of alkaloids, carbohydrates, glycosides, and saponins, suggesting bioactive compounds with potential therapeutic effects. Notably, the extract was negative for proteins, amino acids, reducing sugars, flavonoids, cardiac glycosides, phenolic compounds, and tannins that confirms the plant's explicit bioactive components.
The quantification of phenolic content using a gallic acid standard curve confirms a total phenolic content of 1.896 µg GA/g, which ultimately indicates a moderate level of phenolic compounds in the extract. Similarly, the flavonoid content, determined using a quercetin standard curve, was 260 µg QE/g, that makes Euphorbia microphylla as a significant source of flavonoids.
By focusing protein denaturation, protease release, and membrane stabilisation, the three in vitro methods of anti-inflammatory activity assay together supports the findings Euphorbia microphylla leaf ethanolic extract as core inflammatory pathways. Phytochemicals such as flavonoids and phenolics, which are previously quantified in the extract, highlights on its efficacy, suggesting its therapeutic potential and further interest in formulating standard drugs.