Phytochemical profiling and evaluation of antimicrobial, anti-hemolytic and cytotoxic activities of different parts of Phyllanthus emblica varieties (Anand-1, Banarasi, Chakaiya and Kanchan)

Introduction

One of the most valuable medicinal plants in the Phyllanthaceae family, Phyllanthus emblica L. is widely distributed throughout tropical and subtropical regions including India, Sri Lanka, Malaysia, Thailand and China (Guan et al., 2022) [12] . In India, its cultivation spans several states such as Uttar Pradesh, Gujarat, Madhya Pradesh, Maharashtra and Tamil Nadu with well-known varieties including Chakaiya, Banarasi, Francis, Kanchan, Krishna, Anand-1, Anand-2, NA-7 and NA-10 (Pawar et al., 2024) [24] . Traditionally, P. emblica has been an integral component of Ayurvedic formulations such as triphala and chyawanprash attributed to its rejuvenating and restorative properties (Lim, 2012) [16] . Pharmacological investigations have shown that P. emblica exhibits a broad spectrum of biological activities including antioxidant, anti-inflammatory, antimicrobial, anticancer, hepatoprotective, nephroprotective and antidiabetic effects (Guan et al., 2022; Tiwana et al., 2025) [12,37] . These effects are attributed to its rich phytochemicals profile; phenolics, flavonoids, tannins, alkaloids, terpenoids as well as its high ascorbic acid content (Pareek et al., 2022; Suci and Syahputra, 2024) [23,34] . Earlier studies have mainly emphasized the fruit pulp, yet other parts such as the seed and seedcoat have also been reported to contain significant bioactive compounds like fatty acids, phytosterols and polyphenols suggesting their unexplored medicinal potential (Avinash et al., 2024) [5] .

The qualitative phytochemical screening of solvent extracts is an essential step in identifying the classes of bioactive molecules responsible for these biological activities (Halim et al., 2022) [14] . Also fruits rich in phenolic and flavonoid compounds are known to play a vital role in combating microbial infections and protecting erythrocytes from oxidative damage (Nath and Dhivya, 2019) [21] . Many of these compounds have also garnered significant interest for their potential cytotoxic effects against various cancer cell lines. Therefore, assessing both the antimicrobial and antihemolytic activities of P. emblica extracts can offer insights into its therapeutic relevance. In view of the growing interest in natural remedies as safer alternatives to synthetic drugs, the present study aims to evaluate the phytochemical screening, antimicrobial, antihemolytic and cytotoxic activities of solvent extracts from different parts (pulp, seedcoat and seed) of four Phyllanthus emblica varieties (Chakaiya, Kanchan, Anand-1 and Banarasi). The comparative analysis across multiple solvents provides a better understanding of the extractive efficiency and bioactivity profile of these medicinally significant fruits.

Materials and Methods

Collection of fruits

The fruits of Phyllanthus emblica (Amla) varieties namely Anand-1, Banarasi, Chakaiya and Kanchan were collected from the Agro-forestry Research Station, SDAU, S.K. Nagar, Gujrat.

Phytochemical screening of various part of P. emblica varieties

Phytochemical screening of all P. emblica extracts was carried out to identify the presence of major classes of bioactive compounds.

Test for tannins

One ml of the extract was mixed with 1ml of 5% ferric chloride solution. The formation of a brown-black colour indicated the presence of tannins (Sultana et al., 2009) [35] .

Test for saponins

One ml of the extract was diluted with 2ml of Water and shaken vigorously in a graduated cylinder for 15 minutes. The formation of a persistent layer of foam indicated the presence of saponins (Selvaraj et al. 2014) [30] .

Test for quinones

One ml of the extract was mixed with 1ml of concentrated sulfuric acid. The appearance of a dark red colour indicated the presence of quinones (Singh et al., 2015) [33] .

Test for flavonoids

One ml of the extract was treated with 1ml of 2N sodium hydroxide solution. The development of a yellow colour indicated the presence of flavonoids (Savithramma et al. 2011) [29] .

Test for alkaloids

One ml of the extract was mixed with 2ml of concentrated hydrochloric acid. A few drops of Mayer's reagent were then added. The formation of a green colour indicated the presence of alkaloids (Singh et al., 2015) [33] .

Test for glycosides

One ml of the extract was added to 3ml of chloroform followed by a few drops of 10% ammonium solution. A pink colour formation indicated the presence of glycosides (Zhao et al., 2015) [41] .

Test for terpenoids

One ml of the extract was mixed with 2ml of chloroform and a few drops of concentrated sulfuric acid were added. A reddish-brown colouration at the interface indicated the presence of terpenoids (Savithramma et al. 2011) [29] .

Test for phenols

One ml of the extract was combined with 1ml of Water followed by the addition of a few drops of 10% ferric chloride solution. The development of a dark blue colouration confirmed the presence of phenols (Selvaraj et al. 2014) [30] .

Antimicrobial activity

The antimicrobial activity of various extracts from different parts (pulp, seedcoat and seed) of P. emblica varieties was assessed using the agar well diffusion method with slight modifications as per the procedure described by Manandhar et al. (2019) [17] .

Antibacterial activity

The antibacterial activity of the extracts was evaluated against two Gram-positive (S. aureus, B. cereus) and two Gram-negative (E. coli, S. Typhi) strains using the agar well diffusion method. Fresh bacterial suspensions were adjusted to an OD of 0.1 at 600 nm (Ahmed et al., 2023) [1] and uniformly swabbed (100 µL) onto Mueller-Hinton agar plates. Wells (8 mm) were made with a sterile cork borer and filled with 100 µL of each extract. DMSO and ampicillin served as negative and positive controls, respectively. Plates were kept at 4°C for 1 h to allow diffusion, then incubated at 37°C for 24 h, after which inhibition zones (mm) were measured.

Antifungal activity

Antifungal activity was evaluated against Fusarium oxysporum f. sp. ricini and F. udum using the agar well diffusion method. Spore suspensions were adjusted to an OD of 0.1 at 600 nm (Ahmed et al., 2023) [1] and 100 µL was swabbed onto sterile SDA plates. Wells (8 mm) were filled with 100 µL of each extract; DMSO and nystatin served as negative and positive controls, respectively. Plates were kept at 4°C for 1 h to permit diffusion, then incubated at 28°C for 4-5 days. Antifungal activity was determined by measuring inhibition zone diameters (mm).

Anti-hemolytic assay

The anti-hemolytic activity of Phyllanthus emblica extracts was assessed following Yang et al. (2005) [39] . Fresh human blood was obtained from healthy volunteer via Health Centre, SDAU, Sardarkrushinagar and collected in EDTAtreated tubes. Extracts from four varieties and three fruit parts were tested at concentrations of 125-1000 µg/mL. Fresh human blood (EDTA-treated) was centrifuged at 2000 rpm for 10 min, and erythrocytes were washed with PBS to obtain a 10% hematocrit suspension. For the assay, 250 µL of erythrocyte suspension was mixed with 500 µL of extract (test), PBS (negative control) or water (positive control) and incubated at 37°C for 4 hr. Samples were then diluted to 4.5 mL with PBS, centrifuged and the absorbance of the supernatant was measured at 540 nm (Yang et al., 2017) [40] . Percent hemolysis was calculated as:

Hemolysise rate (%) = [(A extract -A negative control / A positive control ] x 100

Cytotoxic activity (MTT assay)

The cytotoxic activity of methanolic extracts of Phyllanthus emblica was evaluated using the MTT assay against breast cancer (MDA-MB) cell line. Cells were maintained in growth medium with 10% FBS and antibiotics at 37°C in 5% CO₂. After seeding into 96-well plates and allowing 36 hr for adherence, cells were treated with serial dilutions of the extracts (stock = 2 mg/mL). Following incubation, 10 µL of MTT solution was added and plates were further incubated for formazan formation. The medium was removed; crystals were dissolved in 100 µL DMSO and absorbance was read at 570 nm. Cell viability (%) was calculated relative to untreated controls.

Result and Discussion

Phytochemical screening of P. emblica varieties

A comparative phytochemical screening was performed on the pulp, seedcoat and seed of four Phyllanthus emblica varieties using solvents (methanol, ethyl acetate, water and chloroform). Eight major phytochemical groups were assessed (Tables 1 and2). Methanolic pulp extracts showed the richest profile, with abundant phenols and quinones across all varieties and strong flavonoid presence in 'Chakaiya' and 'Kanchan'. Moderate levels of tannins, saponins, alkaloids, glycosides and terpenoids were also detected. Ethyl acetate extracts moderately showed tannins, flavonoids and phenols, whereas aqueous and chloroform extracts showed limited or absence of phytochemicals. In seedcoat and seed, methanol again proved most efficient, yielding notable tannins and quinones along with moderate flavonoids, alkaloids and terpenoids. Other solvents showed poor presence, with chloroform largely inactive. Overall, extraction efficiency was strongly influenced by solvent polarity and plant part, with pulp extracts consistently richest in bioactive constituents. These observations agree with previous studies (Sultana et al., 2009; Singh et al., 2015) [35,33] , which highlight methanol's superior polarity and solubility range and with Rahman et al. (2013) [26] , who reported chloroform's weak performance due to low polarity. The present results confirm methanol as the most effective solvent for broadspectrum phytochemical recovery in P. emblica.

Antimicrobial activity of P. emblica varieties 3.2.1 Antibacterial activity

The present study demonstrated that the antibacterial activity of Phyllanthus emblica extracts strongly depended on the solvent system. Extracts from the pulp, seedcoat, and seed of four varieties were tested against two Gram-positive (S. aureus, B. cereus) and two Gram-negative (E. coli, S. Typhi) strains (Table 3; Fig 1234). The negative control (DMSO) showed no inhibition, while the positive control (Ampicillin) exhibited strong activity (17-29 mm). A clear dependency on solvent polarity was observed for extraction efficacy. aqueous and chloroform extracts were inactive, whereas methanolic and ethyl acetate extracts displayed broad-spectrum antibacterial effects. Methanol consistently yielded the largest inhibition zones, confirming its higher efficiency in extracting active antibacterial constituents. Among plant parts, the pulp showed the strongest activity, followed by the seedcoat and seed. Varietal differences were evident, with Anand-1 showing the highest antibacterial potential. Its seed methanolic extract recorded the maximum inhibition zone (28 ± 1.2 mm) against B. cereus, and the pulp methanolic extract showed high activity against E. coli (26 ± 1.4 mm) and B. cereus (26 ± 1.1 mm). Kanchan and Chakaiya also demonstrated notable inhibition, whereas Banarasi displayed comparatively weak activity. These findings are consistent with earlier reports. The inhibition zones for S. aureus (9-16 mm) align with Anbuselvi and Jha (2015) [2] , while moderate ethyl acetate activity agrees with Goyal et al. (2022) [11] . The methanolic pulp extracts in this study showed stronger E. coli inhibition (up to 26 mm) than the 14.2 mm reported by Anusha (2019) [3] , and comparable to values observed by Ghosh and Dey (2022) [10] . The pronounced inhibition of B. cereus (up to 28 ± 1.2 mm) parallels the results of Phyo et al. (2020) [25] , confirming the potent antibacterial efficacy of P. emblica particularly the methanolic pulp and seed extracts of Anand-1.

The superior performance of Anand-1 is chemically driven by its high concentration of gallic acid, ellagic acid, rutin and quercetin (Tiwana et al., 2025) [38] . Molecular docking studies reveal that ellagic acid and quercetin bind specifically to penicillin-binding protein 3 (PBP-3) and tyrosyl-tRNA synthetase in bacteria (Saini et al., 2024) [28] . This interaction inhibits cell wall synthesis and protein translation, respectively. Furthermore, these phenolics and tannins act by binding to bacterial surface proteins, causing the leakage of intracellular contents and ultimately leading to cell death (Cahyaningrum et al., 2025) [8] .

Antifungal activity of P. emblica varieties

The antifungal efficacy of P. emblica extracts was assessed against Fusarium oxysporum f. sp. ricini and Fusarium udum using the agar well diffusion method. Nystatin served as the positive control and DMSO was the negative control.

Results are presented in Table 4.

A significant finding was that only methanolic pulp extracts exhibited observable antifungal activity. Ethyl acetate, chloroform and water extracts were ineffective, indicating their failure to extract the relevant antifungal constituents. Against F. oxysporum f. sp. ricini, the methanolic pulp extract of the Chakaiya variety showed the highest inhibition (15 mm), followed by Kanchan (14 mm), Banarasi (12 mm) and Anand-1 (5 mm). A similar trend was observed against F. udum, with Chakaiya again showing strong activity (14 mm), followed by Kanchan (12 mm), Banarasi (10 mm) and Anand-1 (9 mm). These results confirm that methanol is an effective solvent for extracting antifungal compounds from P. emblica pulp. The Chakaiya and Kanchan varieties consistently demonstrated superior activity, implying a higher concentration of bioactive antifungal phytochemicals. The inhibition zones observed in this study (ranging from 12-15 mm in effective varieties) align directly with previous research. Arora and Bharti (2014) [4] reported significant activity (12-16 mm) for methanolic amla pulp extracts against Fusarium oxysporum. This efficacy is also supported by findings from Bhalodia et al. (2011) [7] (13.2 mm zone), Goyal et al. (2022) [11] (13-16 mm), and Mazumder et al.

(2008) [18] (11-13 mm) against Fusarium spp., reaffirming the antifungal potency of methanol-extracted pulp components. The significant inhibition zones observed for Chakaiya and Kanchan are attributed to their high levels of hydrolyzable tannins, such as emblicanin A and B (Cárdenas and Laverde 2021) [9] . These tannins function as biological surfactants that compromise fungal cell membrane integrity. This leads to a dose-dependent leakage of water-soluble carbohydrates and intracellular proteins from the fungal hyphae. Transcriptomic profiling suggests that these phytochemicals also down-regulate key metabolic pathways, including the MAPK signalling pathway and porphyrin metabolism, effectively shutting down fungal growth and pathogenicity (Rohilla et al., 2023) [27] .

Anti-hemolytic activity of P. emblica varieties

The anti-hemolytic activity of pulp, seedcoat and seed extracts from four Phyllanthus emblica varieties was assessed using different solvents (Table 5). IC₅₀ values varied notably with both plant part and solvent system. Methanolic pulp extracts showed the strongest activity with the lowest IC₅₀ values (42.30-54.74 µg/mL), followed by ethyl acetate (74.94-151.08 µg/mL), water (131.58-305.13 µg/mL) and chloroform extracts, which were least effective. Across all varieties, pulp extracts consistently outperformed seedcoat and seed extracts, confirming the pulp as the richest source of anti-hemolytic compounds. Overall solvent efficiency followed the order: Methanol > Ethyl acetate > Water > Chloroform, while plant parts followed: Pulp > Seed coat > Seed.

Compared to previous findings by Sharif et al. (2023), who reported an IC₅₀ of 101.08 µg/mL for methanolic fruit extract, the present study recorded much lower IC₅₀ values (42-55 µg/mL), indicating stronger hemolysis inhibition and higher concentrations of active phenolic and flavonoid constituents in the tested materials. The protective effect is chemically linked to Amla's rich phenolic and flavonoid content. Phytochemicals like catechin and epicatechin stabilize the erythrocyte membrane by neutralizing free radicals (Tao et al., 2022) [36] . Furthermore, these compounds specifically regulate the 15lipoxygenase (15-LOX) mediated pathway, preventing the transformation of arachidonic acid into inflammatory mediators that otherwise cause membrane rupture (Saini et al., 2024) [28] .

Cytotoxic activity of P. emblica varieties

The cytotoxic effects of methanolic pulp extract of four Amla varieties were evaluated against a breast cancer cell line (MDA-MB) using an MTT assay. Among the tested varieties, only the Kanchan variety exhibited significant cytotoxic activity. This toxicity was specific, occurring at the 0.5 mg/ml concentration, where it reduced cell viability to approximately 47-56% (Fig 5). In contrast, the other varieties did not show a similar cytotoxic effect. Shashank et al. (2021) [32] reported that methanolic extracts of Amla exhibited cytotoxic effects. Similarly, Baliga et al. (2011) [6] emphasized Amla's broad anticancer potential, noting that its efficacy is strongly influenced by the variety, extraction method and dosage. Amla varieties possess higher levels of bioactive compounds such as polyphenols and tannins, which may account for their enhanced cytotoxic properties. Kanchan's specific efficacy is chemically attributed to its unique accumulation of bioactive polyphenols and tannins. Studies show that Kanchan can contain exceptionally high levels of ellagic acid compared to other cultivars (Zhao et al., 2015) [41] . These components, along with emblicanin A and B, induce apoptosis in MDA-MB-231 cells by modulating the cell cycle at the G2 phase and interfering with cyclin B regulation (Natsakulmongkon and Poeaim 2026) [22] . This results in internucleosomal DNA fragmentation and membrane blebbing specifically in malignant cells, while showing four-fold lower sensitivity toward normal fibroblasts (Huang et al., 2022) [15] .

Conclusion

The present investigation provides a comprehensive comparative assessment of the phytochemical composition and biological efficacy of different parts (pulp, seedcoat and seed) of four Phyllanthus emblica varieties Anand-1, Banarasi, Chakaiya and Kanchan extracted using solvents of varying polarity. The findings collectively highlight the profound influence of solvent type and plant part on the yield and spectrum of bioactive compounds, as well as their associated antimicrobial, antifungal, anti-hemolytic and cytotoxic potentials. Among all tested extracts, the methanolic extracts consistently demonstrated superior extraction efficiency and biological activity, owing to their ability to solubilize a wide range of polar phytochemicals, particularly phenolics, flavonoids, tannins, alkaloids and quinones. The pulp part emerged as the richest source of these bioactive constituents, followed by the seedcoat and seed. This phytochemical abundance was directly reflected in its biological assays. The hierarchy of solvent efficacy (Methanol > Ethyl acetate > Water > Chloroform) and plant part contribution (Pulp > Seedcoat > Seed) was consistent across all analyses, establishing a clear pattern in bioactivity correlation. Anand-1 is scientifically identified as a leader for antibacterial applications due to its flavonoid-PBP-3 binding affinity. Chakaiya is the superior antifungal candidate, acting through tannin-mediated membrane disruption. Most notably, the Kanchan variety stands out as a high-potential source for anticancer molecules, specifically targeting triplenegative breast cancer pathways through G2 cell cycle arrest. This varietal specificity highlights the need for a targeted approach in the development of natural therapeutic formulations and functional foods. Future research should focus on high-resolution metabolomic isolation of these individual compounds to maximize the clinical translation of Phyllanthus emblica in modern healthcare.

Acknowledgement

AcknowledgmentThe author acknowledges the S.D. Agricultural University for providing financial support and conducive working during this study.

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