In-Vitro Evaluation of the Antioxidant and Anti-Inflammatory Activities of the Combined Plant Extracts from Curcuma amada and Phyllanthus emblica

Abstract

This study investigates the in-vitro antioxidant and anti-inflammatory activities of individual and combined ethanol extracts of Curcuma amada Roxb. (mango ginger) and Phyllanthus emblica L. (amla). The plant materials were subjected to maceration extraction using ethanol as solvent. Antioxidant activity was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, while anti-inflammatory activity was assessed through the protein denaturation inhibition assay. The DPPH assay revealed that C. amada extract exhibited potent antioxidant activity with an IC50 value of 440.66 ?g/ml, comparable to the standard ascorbic acid (IC50 = 26.81 ?g/ml). The P. emblica extract demonstrated high antioxidant activity with IC50 of 134.17 ?g/ml. In the protein denaturation inhibition assay, C. amada showed IC50 of >100 ?g/ml, while P. emblica exhibited IC50 > 100 ?g/ml. The combined extract (A+B) displayed IC50 values of 30.69 ?g/ml for DPPH and 88.70 ?g/ml for protein denaturation inhibition. Combination index analysis using the Chou-Talalay method revealed antagonistic interactions between the two extracts at the tested ratios, with CI values exceeding 2.0 at the IC50 level. These findings indicate that while the combination of C. amada and P. emblica ethanol extract possesses high significant antioxidant and moderate anti-inflammatory properties as compared to individual plant extract under the tested conditions.

Keywords

Introduction

   

 

Figure 01 :- Curcuma amada Roxb. & Phyllanthus emblica L.

Chemicals and Reagents

Ethanol (analytical grade, 70% purity) was used as the extraction solvent. 2,2-Diphenyl-1 picrylhydrazyl (DPPH), ascorbic acid, diclofenac sodium, bovine serum albumin (BSA), and phosphate buffered saline (PBS) were obtained from Sigma-Aldrich Chemicals Private Limited (Bangalore, India). All other chemicals and reagents used in this study were of analytical grade and used without further purification. Distilled water was used throughout the experiments.

Extraction Procedure

The powdered plant materials were extracted separately using the maceration technique with 70 % ethanol as the solvent. For each plant material, 50 g of dried powder was placed in an air tight glass container and soaked in 500 ml of ethanol (1:10 w/v ratio). The containers were kept at room temperature with occasional shaking for 72 hours. After the maceration period, the extracts were filtered through Whatman No. 1 filter paper. The marc (residue) was subjected to two additional maceration cycles with fresh ethanol (300 ml each) to ensure exhaustive extraction. The combined filtrates were concentrated under reduced pressure using a rotary evaporator at 40°C. The concentrated extracts were subsequently dried in a electric water bath until constant weight was achieved. The dried extracts were stored in sealed vials at 4°C until further analysis. The percentage yield was calculated using the formula :-

Percentage Yield (%)=Weight of Dried ExtractWeight of Powdered Plant Material×100

   

 

Figure 02 :- Method of Extraction Sample A & B

For combination studies, equal quantities of the dried C. amada and P. emblica extracts were mixed to prepare the combined extract (designated as A+B). Stock solutions of each extract were prepared in the appropriate solvents (methanol for DPPH assay, distilled water for protein denaturation assay) at a concentration of 10 mg/ml and stored at 4°C. Working solutions were prepared fresh on the day of each experiment by appropriate dilution of the stock solutions.

DPPH Radical Scavenging Assay   

The antioxidant activity was evaluated using the DPPH radical scavenging assay following the method described by Brand-Williams et al.^[28] with slight modifications. A 60 μM solution of DPPH was freshly prepared in methanol. For the assay, 200 μl of DPPH solution was mixed with 50 μl of test sample at various concentrations (1.56, 3.12, 6.25, 12.5, 25, 50, 100, 200, 400, 800, and 1000 μg/ml). The mixture was vortexed thoroughly and kept in the dark at room temperature for 15 minutes. The absorbance was measured at 515 nm using a multi-mode microplate reader (MultiSKAN Skyhigh, Thermo Fisher Scientific). Ascorbic acid was used as the reference standard. Control was prepared with DPPH solution only (without any extract or standard), and 95% methanol was used as blank. All determinations were performed in triplicate. The percentage inhibition of DPPH radical was calculated using the formula :-

%Inhibition=Absorbance of Control – Absorbance of TestAbsorbance of Control×100

 

  

 

Figure 03 :- DPPH Radical Scavenging Assay for Standard and Smaple A, B & A+B

Protein Denaturation Inhibition Assay

The anti-inflammatory activity was evaluated using the protein denaturation inhibition assay as described by Mizushima and Kobayashi^[29] with modifications by Sakat et.al.,^[30]. The reaction mixture (0.5 ml) consisted of 0.4 ml of 3% aqueous solution of bovine serum albumin (BSA) and 0.1 ml of test sample at varying concentrations (6.25, 12.5, 25, 50, and 100 μg/ml). The samples were incubated at 37°C for 20 minutes in a water bath. After incubation, 2.5 ml of phosphate buffered saline (pH 6.3) was added to each tube, and the mixtures were heated at 80°C for 10 minutes to induce protein denaturation. After cooling to room temperature, the absorbance was measured at 660 nm using a UV-VIS spectrophotometer (Orion Aquamate 8000, Thermo Scientific). Diclofenac sodium was used as the reference standard. Control was prepared without any test sample. The percentage inhibition of protein denaturation was calculated using the formula :-

%Inhibition=Absorbance of Control – Absorbance of TestAbsorbance of Control×100

    

 

    

 

Figure 04 :- Protein Denaturation Inhibition Assay for Standard and Smaple A, B & A+B

Table 01 :- Percentage Yield of Extracts

Table 03 :- DPPH radical scavenging activity of ethanol extracts (Antioxidant Activity)

Standard	Concentration (μg/ml)	Absorbance at 515nm			Triplicate Average	Percentage Inhibition (%)
		OD1	OD2	OD3
Control	-	0.866	0.863	0.863	0.864	0.864
Ascorbic acid	1.56	0.833	0.831	0.830	0.831	3.94
	3.12	0.763	0.767	0.761	0.764	11.92
	6.25	0.719	0.713	0.712	0.715	17.59
	12.5	0.581	0.583	0.580	0.581	32.87
	25	0.481	0.485	0.480	0.482	44.44
	50	0.155	0.157	0.154	0.155	82.18
	100	0.093	0.092	0.093	0.093	89.28
	200	0.085	0.084	0.084	0.084	90.23
	400	0.065	0.065	0.065	0.065	92.48
	800	0.054	0.057	0.055	0.055	93.63
	1000	0.041	0.042	0.042	0.041	95.20
	IC 50					26.81μg/ml

 

Sample code	Concentration (μg/ml)	Absorbance at 515nm			Triplicate Average	Percentage Inhibition (%)
		OD1	OD2	OD3
Control	-	0.872	0.873	0.870	0.872	-
Sample A Phyllanthus emblica	1.56	0.838	0.837	0.831	0.835	4.20
	3.12	0.816	0.815	0.818	0.816	6.38
	6.25	0.787	0.786	0.789	0.787	9.71
	12.5	0.750	0.748	0.746	0.748	14.22
	25	0.658	0.658	0.654	0.657	24.69
	50	0.572	0.568	0.572	0.571	34.56
	100	0.483	0.481	0.483	0.482	44.69
	200	0.336	0.334	0.339	0.336	61.43
	400	0.216	0.213	0.215	0.215	75.38
	800	0.093	0.091	0.092	0.092	89.47
	1000	0.091	0.093	0.090	0.091	89.53
	IC 50					134.17 μg/ml
		0.871	0.873	0.870	0.871	-
Sample B Curcuma amada	1.56	0.851	0.847	0.848	0.849	2.56
	3.12	0.829	0.828	0.826	0.828	4.98
	6.25	0.792	0.799	0.793	0.795	8.76
	12.5	0.764	0.764	0.762	0.763	12.36
	25	0.725	0.728	0.726	0.726	16.61
	50	0.689	0.687	0.687	0.688	21.05
	100	0.619	0.613	0.611	0.614	29.47
	200	0.537	0.536	0.534	0.536	38.50
	400	0.454	0.456	0.451	0.454	47.91
	800	0.285	0.285	0.282	0.284	67.39
	1000	0.164	0.161	0.161	0.162	81.40
	IC 50					440.66 μg/ml
		0.869	0.873	0.873	0.872	-
Sample A+B	1.56	0.824	0.822	0.827	0.824	5.47
	3.12	0.793	0.795	0.791	0.793	9.06
	6.25	0.739	0.734	0.736	0.736	15.56
	12.5	0.654	0.653	0.658	0.655	24.89
	25	0.499	0.502	0.503	0.501	42.50
	50	0.207	0.206	0.206	0.206	76.34
	100	0.109	0.109	0.109	0.109	87.50
	200	0.106	0.107	0.104	0.106	87.88
	400	0.100	0.102	0.100	0.101	88.47
	800	0.099	0.099	0.096	0.098	88.76
	1000	0.090	0.094	0.092	0.092	89.44
	IC 50					30.69 μg/ml

 

Figure 05 :- DPPH radical scavenging activity of ethanol extracts (Antioxidant Activity)

Table 04 :- Protein denaturation inhibition activity of ethanol extracts (Anti-inflammatory Activity)

Standard	Concentration (μg/ml)	Absorbance at 660 nm	Percentage Inhibition (%)
Control	-	0.857	-
Diclofenac Sodium	6.25	0.790	7.82
	12.5	0.713	16.80
	25	0.592	30.92
	50	0.401	53.21
	100	0.121	85.88
	IC 50		46.21μg/ml

 

Sample code	Concentration (μg/ml)	Absorbance at 660 nm	Percentage Inhibition (%)
Control	-	0.845	-
Sample A Phyllanthus emblica	6.25	0.840	0.59
	12.5	0.782	7.46
	25	0.712	15.74
	50	0.594	29.70
	100	0.506	40.12
	IC 50*		>100μg/ml
Sample B Curcuma amada	6.25	0.811	4.02
	12.5	0.776	8.17
	25	0.727	13.96
	50	0.675	20.12
	100	0.566	33.02
	IC 50*		>100μg/ml
Sample A+B	6.25	0.679	19.64
	12.5	0.597	29.35
	25	0.531	37.16
	50	0.583	31.01
	100	0.405	52.07
	IC 50*		88.70μg/ml

Figure 06 :- Protein denaturation inhibition activity of ethanol extracts (Anti-inflammatory Activity)

Table 05 :- Combination index analysis for DPPH and protein denaturation assays.

Assay	IC50 A (μg/ml)	IC50 B (μg/ml)	IC50 A+B (μg/ml)	CI Value	Interpretation
DPPH	134.17	440.66	30.69	0.15	Synergism
Protein Denaturation	>100	>100	88.70	> 0.89	Synergism

Figure 07 :- IC₅₀ Comparison for DPPH and Protein denaturation assays (Sample A, B & A+B)

DISCUSSION

The present study evaluated the antioxidant and anti-inflammatory activities of individual and combined ethanol extracts of Curcuma amada and Phyllanthus emblica using in-vitro assays. The findings revealed that C. amada ethanol extract possesses potent antioxidant activity, as evidenced by its low IC50 value (440.66 μg/ml) in the DPPH assay, which was comparable to the reference standard ascorbic acid (26.81 μg/ml). This potent radical scavenging activity can be attributed to the presence of curcuminoids, particularly curcumin, which is well-documented for its antioxidant properties^[31,32]. Curcumin and its derivatives exert antioxidant effects through multiple mechanisms, including direct free radical scavenging, metal ion chelation, and upregulation of endogenous antioxidant enzymes such as superoxide dismutase and catalase^[33].

The moderate DPPH radical scavenging activity observed for P. emblica extract (IC50 = 134.17 μg/ml) was somewhat expected given the well-documented antioxidant properties of this plant^[20,21]. However, it is important to consider that the extraction method and solvent significantly influence the phytochemical profile of the resulting extract. Ethanol, while effective for extracting a broad range of polar and semi-polar compounds, may not be optimal for extracting the highly water-soluble vitamin C, which is a major antioxidant constituent of P. emblica. Furthermore, the antioxidant activity of P. emblica has been shown to be highly dependent on the geographical origin, harvest time, and post-harvest processing methods^[34]. The tannin-rich fraction of amla, particularly emblicanin A and B, has been reported to possess superior antioxidant activity compared to other fractions^[35], and these compounds may have been completely extracted under the conditions employed in this study.

The protein denaturation inhibition assay revealed that C. amada extract possesses moderate anti-inflammatory activity (IC50 = > 100 μg/ml), while P. emblica showed relatively weak activity (IC50 > 100 μg/ml). The anti-inflammatory effect of C. amada can be attributed to its curcuminoid content, which has been extensively studied for its ability to modulate inflammatory pathways^[36]. Curcumin suppresses the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), inhibits cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), and blocks nuclear factor-kappa B (NF-κB) signaling^[37,38]. The protein denaturation inhibition mechanism is particularly relevant to the management of rheumatoid arthritis, where autoantigens produced through protein denaturation trigger immune responses leading to joint inflammation and damage^[39].

A critical finding of this study was the synergistic interaction observed between the two extracts when combined. The combination index (CI) analysis was performed to quantitatively assess the nature of interaction between C. amada and P. emblica extracts. For the DPPH assay at the IC50 level, with the combination prepared in a 1:1 ratio, the calculated CI value was 0.15 and For the Protein denaturation assay at the IC50 level, with the combination prepared in a 1:1 ratio, the calculated CI value was 0.89 (Table 05). This value significantly indicating synergistic interaction between the two extracts. First, the substantial difference in potency between the two extracts, with P. embllica being considerably more active than C. amada, means that combining them effectively enhance the active constituents of C. amada. Second, phytochemical interactions between constituents of the two extracts may interfere with the bioavailability or activity of key compounds. For instance, tannins from P. emblica may form complexes with curcuminoids, potentially enhancing their availability for radical scavenging or protein stabilization^[40]. Third, the 1:1 ratio employed in this study may represent the highest optimal ratio for synergy; different ratios could potentially yield different interaction outcomes.

The finding of synergistic interaction contrasts with some previous reports of synergistic antioxidant effects in polyherbal formulations containing these or related plants^[41,42]. However, direct comparison is challenging due to differences in extraction methods, ratio of constituents, and assay conditions. It is also noteworthy that in vitro assays, while providing valuable initial screening data, may not fully predict in vivo efficacy due to factors such as bioavailability, metabolism, and tissue distribution. The demonstration of synergism in this study highlights the importance of systematic combination studies using quantitative methods such as the Chou-Talalay CI analysis before advocating for polyherbal combinations.

The extraction method employed in this study, maceration with ethanol, represents a traditional and widely used approach that yields a broad spectrum of phytoconstituents. The yield obtained for C. amada (30%) was consistent with previous reports using similar extraction protocols^[43]. The higher yield for P. emblica (50%) reflects the rich content of extractable constituents in amla fruits. Future studies may consider employing sequential extraction with solvents of varying polarity to obtain fractionated extracts and identify the most bioactive fractions. Additionally, exploring alternative ratios for the combination, employing response surface methodology to identify optimal proportions, and conducting in vivo validation studies would provide valuable insights for potential therapeutic applications.

CONCLUSION

This study evaluated the antioxidant and anti-inflammatory activities of ethanol extracts of Curcuma amada and Phyllanthus emblica, individually and in combination. The results demonstrated that C. amada ethanol extract possesses potent DPPH radical scavenging activity (IC50 = 440.66 μg/ml) and moderate protein denaturation inhibitory activity (IC50 = >100 μg/ml). In contrast, P. emblica ethanol extract exhibited stronger activities in DPPH radical scavenging activity (IC50 = 134.17 μg/ml) and moderate protein denaturation inhibitory activity (IC50 = >100 μg/ml). The combination of the two extracts produce highest synergistic enhancement; rather, individual index analysis revealed antagonistic interactions in both the DPPH assay and protein denaturation inhibition assay. These findings best score the importance of systematic combination studies using quantitative analytical methods and suggest that the combination of these two plants in a 1:1 ratio for achieving highest enhanced antioxidant or anti-inflammatory effects.

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