Central Composite Design based Optimization of Synergistic Anti-cholesterol activity in Garcinia indica and Thespesia populnea extracts

 

K B Amrutha Rathod, Roopashree T S

Government College of Pharmacy, Bangalore.

 

ABSTRACT:

In this study, the anti-cholesterol potential of methanolic extracts of Garcinia indica bark and Thespesia populnea leaves was investigated using the Liebermann-Burchard colorimetric assay. Both individual and combined extract effects were examined; with the optimization of concentrations achieved through Central Composite Design [CCD]. The principal aim of this investigation was to evaluate the cholesterol-inhibitory characteristics of these extracts and ascertain their possible application as natural therapeutics for the regulation of cholesterol. The methanolic extracts of Garcinia indica and Thespesia populnea demonstrated significant cholesterol inhibition. Garcinia indica showed an inhibition rate of 76.43±0.39%, while Thespesia populnea displayed an inhibition rate of 71.83±0.46% at a concentration of 80 µg/mL. Thespesia populnea and Garcinia indica both had significant anti-cholesterol action, as indicated by their respective IC50 values [18.25 µg/mL and 16.43 µg/mL], which represent the concentration needed to prevent 50% of cholesterol development. Further, optimization using CCD identified that a combination of 63.79 µg/mL of Garcinia indica and 57.86 µg/mL of Thespesia populnea achieved an enhanced cholesterol inhibition rate of 81.49%, suggesting a synergistic effect when both extracts were combined.  GraphPad Prism software was used to conduct a statistical study utilizing one-way ANOVA and Dunnett's test to determine the significance of the extracts' effects relative to the standard. Using Design Expert® Software, the CCD optimization yielded important information on the ideal concentrations needed for these plant extracts to maximally suppress cholesterol.  The findings from this study suggest that the combined extracts of Garcinia indica and Thespesia populnea offer a promising natural alternative for cholesterol regulation and the prevention of cardiovascular diseases. These findings highlight the plant extracts' potential as natural herbal remedies for controlling cholesterol. Further research is recommended to elucidate the mechanistic pathways of action, as well as to explore the clinical applications of these extracts, with the ultimate goal of validating their efficacy and safety for use in broader contexts of cholesterol management and cardiovascular disease prevention.

 

 

 

INTRODUCTION:

Heart attacks and strokes are among the world's leading causes of death, and they are largely caused by cardiovascular diseases, which are facilitated by elevated cholesterol levels¹. increased amounts of low-density lipoprotein [LDL], the cholesterol commonly referred to as "bad cholesterol," are associated with atherosclerosis —an accumulation of fatty deposits in the arteries that impedes blood flow and heightens the risk of severe cardiovascular events². To combat this, a variety of strategies are employed, including pharmacological treatments and lifestyle adjustments^3,4. Although pharmacological drugs such as fibrates, bile acid sequestrants, cholesterol absorption inhibitors, and statins are frequently used to control cholesterol levels, non-pharmacological methods such as dietary modifications and natural supplements are getting progressively more common^5,6.

 

Medicinal plants offer a promising avenue for natural cholesterol management. Among these, Garcinia indica⁷. [GI] and Thespesia populnea [TP] have garnered attention for their potential anti-cholesterol properties. Garcinia indica [Clusiaceae]⁸ bark contains bioactive compounds such as garcinol, hydroxycitric acid [HCA], and xanthones, which have been reported to influence cholesterol metabolism. Similarly, Thespesia populnea [Malvaceae]^9,10 leaves are rich in phytochemicals including flavonoids, tannins, and terpenoids, suggesting potential cholesterol-modulating effects^11-13.

 

This study seeks to evaluate the anti-cholesterol characteristics of Garcinia indica bark and Thespesia populnea leaves in light of the increasing prevalence of hypercholesterolemia and the need for safe and effective therapeutic options. By employing in-vitro models, the study seeks to elucidate the potential of these natural extracts as viable alternatives for managing cholesterol levels and improving cardiovascular health.

 

MATERIALS AND METHODS:

Materials and Methods

Collection and authentication of Garcinia indica bark and Thespesia populnea leaves: Bark of Garcinia indica and leaves of Thespesia populnea were collected in December 2023 from Kundapur, Udupi district, Mangalore, Karnataka, and Bengaluru, respectively. On June 23, 2024, Dr. V. Rama Rao, Research Officer of the Central Ayurveda Research Institute, Uttarahalli, Bengaluru, verified the authenticity of the plant sample. Following collection, the plant parts were air-dried in a shaded area for several weeks. Subsequently, they were coarsely ground using a laboratory mixer, sieved through a #24 sieve, and kept for later use in sealed containers.

 

Successive solvent extraction of crude drugs: Using a Soxhlet extractor, powdered plant material was sequentially extracted with solvents [petroleum ether, chloroform, ethyl acetate, and methanol] in increasing polarity. Each solvent was heated, condensed, and passed through the plant material repeatedly until complete extraction was achieved. The plant material was thoroughly dried before applying each higher-polarity solvent. The resulting extracts were concentrated under low temperature conditions, air-dried, and their yields and properties were recorded for further analysis¹⁴.

Assessment of the anti-cholesterol action in vitro:

A colorimetric technique called Liebermann-Burchard was used to assess the extracts anti-cholesterol efficacy.

 

Preparation of Solutions:

Cholesterol Solution: At 45°C in a water bath, 25 mg of cholesterol were dissolved in 10 ml of ethanol to create a concentration of 2.5 mg/ml cholesterol solution.

Standard and Sample Solutions: Following a 100 μg/ml ethanolic dissolution of the extracts or atorvastatin, the solutions were serially diluted to yield eventual concentrations of 20,40,60, and 80 μg/ml.

Liebermann-Burchard Reagent: A chilled sulfuric acid-acetic anhydride mixture was prepared in a refrigerated amber-colored bottle. The mixture consisted of 5 ml of concentrated sulfuric acid and 50 ml of chilled acetic anhydride¹⁵.

Procedure: 0.4 ml of each extract at different concentrations was placed to a of 25 ml volumetric flasks. Afterward, 2 ml of Liebermann-Burchard reagent was slowly added, following the addition of one ml of the 2.5 mg per ml cholesterol mixture to each flask. A quarter of an hour was provided to the mixtures to react. Each solution's absorbance was measured at a wavelength of 440 nm using UV spectroscopy, with ethanol as the blank. One ml of cholesterol solution, two ml of Liebermann-Burchard reagent, and 0.4 ml of ethanol were combined to create the control flask. For the atorvastatin standard solution, the same process was used^16,17. The percentage of cholesterol inhibition was calculated using the following formula¹⁸:

% Inhibition of Cholesterol = [Control absorbance – Sample absorbance] / Control absorbance x 100

 

Optimization and Statistical Analysis:

Central Composite Design (CCD)^19,20. In order to assess potential synergistic effects and improve the extracts' overall anti-cholesterol efficacy, a Central Composite Design [CCD] was utilized. Thespesia populnea [B] and Garcinia indica [A] methanolic extracts were used as independent variables, while the percentage inhibition [R1] was the response variable. The optimization process utilized Design Expert® Software [version 11], with extract concentrations ranging from 20 to 80 µg/mL as the low and high values, respectively. The desirability function was utilized in numerical optimization to determine the ideal parameters for achieving the highest level of anti-cholesterol activity.

 

Statistical Analysis:

GraphPad Prism [version 10] was used to calculate the IC50 values for the anti-cholesterol activity and to evaluate the statistical significance between the samples and the standard using one-way ANOVA and Dunnett's multiple comparison test. CCD in Design Expert Software was used to carry out the optimization analysis.²¹

RESULT:

Successive solvent extraction of crude drugs: Successive solvent extraction of Garcinia indica bark and Thespesia populnea leaves was conducted using a Soxhlet apparatus. Methanol proved to be the most effective solvent, resulting in the highest yield of 6.6% for Garcinia indica and 3.92% for Thespesia populnea.

 

Liebermann-Burchard (LB) colorimetric assay's in vitro anti-cholesterol activity:

The plant extracts' ability to lower cholesterol levels was evaluated by the use of the Liebermann-Burchard colorimetric assay. One-way ANOVA revealed significant differences among the extracts [Figures 1 and 2]. The IC50 values indicated varying degrees of cholesterol inhibition, with the methanolic extracts of Garcinia indica and Thespesia populnea showing the most notable activity [Table 1].

 

 

Figure 1. Percentage of cholesterol inhibition by Garcinia indica [GI] extracts at various concentrations, including pet Ether [PE], chloroform [C], ethyl acetate [EA] and methanol [MET]. The results are presented as mean±SD [n=3]. Statistical significance was determined using one-way ANOVA with Dunnett’s test, with p-values categorized as 0.033[*], 0.002 [**], <0.001[***].

 

 

Figure 2. Percentage of cholesterol inhibition by Thespesia populnea [TP] extracts at various concentrations, including pet ether [PE], chloroform [C], ethyl Acetate [EA] and methanol [MET]. The results are presented as mean±SD [n=3]. Statistical significance was determined using one-way ANOVA with Dunnett’s test, with p-values categorized as 0.033[*], 0.002 [**], <0.001[***].

Table  1. In-vitro anti-cholesterol effect of Garcinia indica and Thespesia populnea successive extracts.

Extracts/ Standards	IC50
	Garcinia indica (µg/mL)	Thespesia populnea (µg/mL)
Petroleum ether extract	26.74	31.06
Chloroform Extract	21.94	24.24
Ethyl acetate extract	19.34	23.38
Methanol extract	16.43	18.25
Atorvastatin	13.68

 

Anti-Cholesterol Activity of Individual and Combined Extracts:

Individual plant extracts:

The anti-cholesterol activity of Garcinia indica and Thespesia populnea extracts was evaluated. Among the different extracts, the methanolic extract of Garcinia indica exhibited the highest cholesterol inhibition at 76.43% at 80 µg/mL, while the methanolic extract of Thespesia populnea showed 71.83% inhibition at the same concentration. These results suggest both plants possess notable anti-cholesterol properties, with Garcinia indica showing efficacy close to that of the standard drug atorvastatin.

 

Anti-cholesterol activity optimization of combined extracts:

The cholesterol inhibition values were optimized using the Central Composite Design model for the combined methanolic extracts of Garcinia indica and Thespesia populnea. The results of the CCD optimization are presented in Table 2. The highest cholesterol inhibition [86.767%] was observed when both extracts were used at 80 µg/mL. The lowest inhibition [61.423%] occurred at lower concentrations of Garcinia indica [7.57 µg/mL].

 

Table 2 Optimization of combined methanolic extracts using CCD.

Standard	Run	Factor 1 A: µg/mL of Garcinia indica	Factor 2 B: µg/mL of Thespesia populnea	Response 1 % Cholesterol inhibition
6	1	80	80	86.767
10	2	50	50	75.5
11	3	92.42641	50	76.42
1	4	7.573593	50	61.423
3	5	50	50	65.911
9	6	20	20	78.42
8	7	50	50	78.55
2	8	80	20	74.17
12	9	50	50	78.64
13	10	50	92.42641	79.42
5	11	50	7.573593	63.192
7	12	20	80	72.388
4	13	50	50	82.855

 

ANOVA and Model Fit:

Table 3 displays the results of the analysis of variance [ANOVA] used to determine the model's statistical significance. Thespesia populnea [Factor B] and Garcinia indica [Factor A] were discovered to have a substantial effect on cholesterol inhibition, with the model being determined to be significant [p < 0.05]. However, the interaction term [AB] and quadratic term for Thespesia populnea [B²] were not statistically significant.

 

Table 3 ANOVA for Quadratic model (Response 1: % cholesterol inhibition)

Source	Sum of Squares	df	Mean Square	F- value	P- value
Model	624.09	5	124.82	17.77	0.0007	Significant
A- Garcinia indica	496.61	1	496.61	70.69	< 0.0001
B- Thespesia populnea	59.88	1	59.88	8.52	0.0224
AB	4.40	1	4.40	0.6269	0.4545
A²	39.53	1	39.53	5.63	0.0494
B²	31.83	1	31.83	4.53	0.0708
Residual	49.17	7	7.02
Lack of Fit	38.34	3	12.78	4.72	0.0841	Not significant
Pure Error	10.84	4	2.71
Cor Total	673.26	12

 

The F-value of 17.77 and P-value of 0.0007 for the overall model indicate that the model is highly significant. The linear term for Garcinia indica [A] was particularly significant [p<0.0001], while Thespesia populnea [B] also played a role [p = 0.0224].

 

Table 4 presents model fit statistics that indicate an adequate model with a R² of 0.9270, meaning that the model accounts for about 92.7% of the variation in cholesterol inhibition. The adjusted R² value of 0.8748 further supports the robustness of the model.

 

Table 4 Fit Data

Std. Dev.	2.65	R²	0.9270
Mean	74.90	Adjusted R²	0.8748
C.V. %	3.54	Predicted R²	0.5699
		Adeq Precision	12.8018

 

Model Equation, Response Surface Plots, and Contour Plot:

The quadratic model for predicting cholesterol inhibition is provided below, showing how the concentrations of Garcinia indica and Thespesia populnea influence cholesterol inhibition:

 

% Cholesterol Inhibition=77.68 + 7.88A + 2.74B + 1.05AB - 2.38A² - 2.14B².

 

This equation suggests that increasing the concentration of Garcinia indica has a more pronounced effect on cholesterol inhibition compared to Thespesia populnea. The interaction between the two factors [AB] and the quadratic terms, particularly for Thespesia populnea, did not significantly contribute to cholesterol inhibition.

 

The response surface plots [Figure 3] and contour plot [Figure 4] illustrate the combined effects of the extracts on cholesterol inhibition, with the maximum inhibition observed at higher concentrations of both extracts. The contour plot provides a visual representation of the optimal combinations of Garcinia indica and Thespesia populnea for achieving the highest cholesterol inhibition.

Figure 3. Response surface plot showing % cholesterol inhibition by methanolic extracts of Garcinia indica and Thespesia populnea [3D Surface].

 

 

 

 

 

 

 

 

 

Figure 4. Contour plot illustrating the combined effect of all factors on cholesterol inhibition by methanolic extracts of Garcinia indica and Thespesia populnea.

 

Figure 5. Contour plot showing the interaction between methanolic of Garcinia indica and Thespesia populnea.

 

Optimization Using Desirability Function:

The desirability function method was applied, and the optimal experimental conditions resulted in 81.49% cholesterol inhibition, as shown in Table 5.

 

Table 5 Solutions from Numerical Optimization

Garcinia indica (µg/mL)	Thespesia populnea (µg/mL)	% Cholesterol Inhibition	Desirability
63.79	57.86	81.49	1	Selected
68.34	46.67	81.20	1
72.24	35.90	80.09	1
75.73	46.44	82.22	1
76.15	42.77	81.73	1

 

DISCUSSION:

The present study underscores the significant anti-cholesterol potential of Garcinia indica and Thespesia populnea extracts, particularly their methanolic extracts, which demonstrated superior cholesterol inhibition. Notably, the methanolic extract of Garcinia indica exhibited a cholesterol inhibition rate of 76.43% at 80 µg/mL, while the methanolic extract of Thespesia populnea showed a 71.83% inhibition rate at the same concentration. These rates closely mirror the inhibition rate of atorvastatin [78%], suggesting their potential as viable alternatives to synthetic drugs.

 

Central Composite Design optimization further revealed the synergistic effects of combining the extracts. The optimal combination of Garcinia indica and Thespesia populnea resulted in a notable enhancement of cholesterol inhibition to 81.49%, supporting the hypothesis that lower doses of combined extracts can achieve substantial therapeutic effects, potentially minimizing side effects associated with higher doses of individual agents.

 

The statistical robustness of the model, as evidenced by the high R² value of 0.9270 and the derived equation, emphasizes the influence of both extracts on cholesterol inhibition. The positive linear terms indicate enhanced cholesterol inhibition, while the negative quadratic terms highlight diminishing effects at higher concentrations, reinforcing the importance of precise dosage in maximizing efficacy.

 

Furthermore, this study aligns with existing literature on the benefits of polyherbal formulations, which often outperform single-agent therapies due to interactions between bioactive compounds. The synergistic combination of Garcinia indica and Thespesia populnea methanolic extracts presents a promising avenue for developing novel cholesterol-lowering therapies. Future research should focus on clinical trials and further exploration of the underlying mechanisms behind these synergistic effects.

 

CONCLUSION:

The results of this investigation show that methanolic extracts from Thespesia populnea and Garcinia indica have encouraging anti-cholesterol properties. The synergistic effects observed when these extracts are combined suggest a promising avenue for developing novel natural therapies for cholesterol management. The results underscore the importance of further research to validate the efficacy and safety of these extracts for broader applications in cardiovascular disease prevention. Additionally, future studies should delve deeper into the underlying mechanisms of action to optimize their therapeutic potential.

 

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Received on 20.09.2024      Revised on 11.01.2025 Accepted on 19.04.2025      Published on 10.07.2025 Available online from July 17, 2025 Asian J. Pharm. Res. 2025; 15(3):249-254. DOI: 10.52711/2231-5691.2025.00040 ©Asian Pharma Press All Right Reserved
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