Optimization of Extraction Process for Kaempferia Parviflora in Vietnam Using Response Surface Methodology

Abstract

Objective

Kaempferia parviflora collected in the Bay Nui region of An Giang, Vietnam. This project aims to build an extraction process with optimized parameters using the RSM model to improve the quality and polyphenol and flavonoid content of extracts from KP.

Method

Kaempferia parviflora was extracted using the ultrasound method, with factors such as temperature, time, and solvent volume affecting the process. The biological activities were assessed using several methods, including the DPPH assay, ABTS assay, agar disk-diffusion method, and the Minimum Inhibitory Concentration (MIC) test.

Results

The content of polyphenols and flavonoids was evaluated to base on the RSM with the optimal parameters from the model (at 96 min, volume 15.4 mL, temperature 66 ^oC) of 81.57 (mgGAE).(gDW)⁻¹ and 50.70 (mgQE).(gDW)⁻¹ (R²= 0.999 and 0.993 > 0.75). The antioxidant capacity of the extract was found to be high with DPPH and ABTS results of 6.990 and 0.014 mg.mL⁻¹. Besides, the antibacterial ability of the sample was also recorded at the minimum inhibitory concentration of Escherichia coli of 10 mg.mL⁻¹ and Staphylococcus aureus of 15 mg.mL⁻¹.

Conclusions

The study showed that the ultrasonic extraction method has shown superior time-saving ability. The extraction conditions were optimized to ensure high flavonoid and polyphenol content, with good activity. It can be seen that Kaempferia parviflora extract had great potential for application in the medical field.

Keywords

response surface methodology optimization extraction process ultrasound method

Introduction

In recent years, natural compounds with antioxidant and antimicrobial activities with high efficiency, low toxicity and reliability were chosen by many people and have high applicability. Kaempferia parviflora (KP) belongs to the Zingiberaceae family and has been used for a long time in traditional medicine to promote health.¹ They have been studied extensively for their pharmacological and chemical effects. Recent studies show that KP rhizome has anti-malarial, anti-viral, anti-bacterial, anti-gastric and anti-fungal effects.^2-6 In traditional medicine, decoction of KP rhizome was used to treat fungal infections, impotence, allergies, asthma, gout, diarrhea, dysentery, stomach ulcers and diabetes.^4,6-9 Among them, the main compounds founded in KP include polyphenols, flavonoids, glycosides, and steroids.^4,5,7,10 Among them, the main group was flavonoids with the main component being 7-methoxy derivatives.^11-13

Vietnam was known as a country of diverse medicinal plants with favorable climate and soil conditions. KP was also widely grown and used in traditional medicine.^4,14 To be able to exploit the value of the potential of KP, extraction was one of the important processes to obtain biological compounds.^7,15-17 Polyphenols and flavonoids in the extraction process could be affected by many different factors. In particular, solvent, time, temperature, material/solvent ratio were considered the main factors.^18,19 In this situation, where there were many variables affecting the extraction process, Response Surface Methodology (RSM) was a suitable technique to optimize the extraction process. In RSM, Box-Behnken design and Central composite design were among the most popular models for determining optimal conditions. It was an important alternative to avoid time-consuming experiments. Therefore, the RSM Model was widely applied in processes that optimize parameters or conditions that affect processes.^20-22 Therefore, to clarify the scientific basis and value of using KP collected in the Seven Mountains region of An Giang. This project aimed to build an extraction process with optimized parameters using the RSM model to improve the quality and polyphenol and flavonoid content of extracts from KP.

Experimental

Pretreatment and Chemicals

Folin & Ciocalteu's phenol reagent (2N), DPPH (2,2-Diphenyl-1-picrylhydrazyl, ≥ 95%), ABTS (2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, ≥ 98%), L-Ascorbic acid (Vitamin C, ≥ 99%), Amoxicillin were provided by Sigma-Aldrich, America. Sodium Carbonate (Na₂CO₃), Aluminum chloride (AlCl₃), Potassium acetate (CH₃COOK), Methanol (CH₃OH), Potassium peroxydisulfate (K₂S₂O₈), Sodium Chloride (NaCl) were provided by Xilong, China. Peptone powder; Yeast Extract Powder; Agar powder were provided by Himedia, India. Bacterial strains including Staphylococcus aureus (S. aureus) NRRL B-313, Escherichia coli (E. coli) NRRL B-409 were preserved on Luria - Bertani (LB) agar blocks at 4 °C. KP rhizomes were collected in Bay Nui area, An Giang, Vietnam during April and May of the year. It was cut into 20–30 mm pieces, drying hot air oven at 65 °C for 24 h, ground into fine powder, and stored in a desiccator at room temperature.

Ultrasonic Extraction Method

KP extract was extracted by ultrasonic method based on Chaisuwan's research.¹⁷ 1 gram of KP powder was extracted with 10–20 mL of 70% Ethanol solvent for 30–90 min at 50–70 °C using an Ultrasonic bath - Pro 100S (Frequency 40 kHz, power 360 W, made in Slovenia) . Then, the extract was filtered through filter paper. The extraction process was performed 2 times. Samples after filtration were stored in a 10 °C refrigerator.

Method for Determining Total Polyphenol Content

Total phenolic content was determined through the Folin – Ciocalteu method.¹⁰ 0.5 mL of extract or standard gallic acid solution (with concentration from 0.05 to 3 mg.mL⁻¹) added to 2.5 mL of Folin – Ciocalteu (10%) and shake well. After 4 min, 2 mL of Na₂CO₃ solution was added to the mixture, shaken well and incubated for 2 h at room temperature. The absorbance of the solution after reaction was measured at 760 nm. Gallic acid was used as a reference standard and the results were equivalent to milligrams of gallic acid/1 gram of the sample's dry weight ((mgGAE).(gDW)⁻¹).

Method for Determining Flavonoid Content

Total flavonoid content was determined based on the Sitthichai method.¹⁰ 0.5 mL of the sample solution (diluted to the appropriate concentration) was added to 0.1 mL of 10% AlCl₃ solution. 0.1 mL of 1 M CH₃COOK solution and 4.3 mL of distilled water were added to the mixture and shaken well. Leave the solution at room temperature for 30 min. Then measure the absorbance at 415 nm on a UV-Vis spectrophotometer. Quercetin was used as a reference standard and the results were equivalent to milligrams of Quercetin /1 gram of the sample's dry weight (mgQE).(gDW)⁻¹).

Method to Implement RSM

The parameters influencing the extraction process were optimized using the RSM with a Box-Behnken Design (BBD).²³ The extract of KP was assessed based on its total polyphenol and flavonoid content. The model's evaluation factors were categorized into three levels: central variable (0), low level (−1), and high level (+1). Analysis of Variance (ANOVA) was performed using Design-Expert software (version 13, State Ease, Minneapolis, USA). The ANOVA of the quadratic linear regression model was employed to analyze the impact of input and output variables, as well as the correlation between response functions and independent variables. The total number of experiments for the BBD matrix was determined using the appropriate formula.²⁰

N = 2 k \times (k - 1) + c = 2 \times 3 \times (3 - 1) + 5 = 17

(1)

In which: k is the number of independent variables, N is the total number of experiments and c is the number of times the central experiment is repeated.

The experiment was performed according to the experimental matrix shown in Table 1. The results of the experiments were listed in Table 1. The response value of adsorption efficiency (Y), and the mathematical model were represented by the quadratic regression equation²⁴:

Y = β_{o} + \sum_{i = 1}^{k} β_{i} x_{i} + \sum_{i = 1}^{k} β_{i i} x_{i}^{2} + \sum_{1 < i < j}^{k} β_{i j} x_{i} x_{j} + ε

(2)

Table 1.

Matrix of Independent Variables and Levels.

Number	Independent Factor	Symbol	Mức
Number	Independent Factor	Symbol	−1	0	+1
1	Extraction time (min)	X₁	60	90	120
2	Temperature (°C)	X₂	10	15	20
3	Solvent volume (mL)	X₃	40	60	80

In which: Y is the theoretical adsorption efficiency, x_i is the independent variables, β is the model constant, βi is the linear coefficient, βii is the quadratic coefficient, βij is the correlation coefficient.

From the values of the X variables and applying the second-order polynomial regression equation, 17 independent experiments were designed. In ANOVA, evaluating the fitness of the model done by studying the dispersion of the data set. To evaluate specifically, the model would be evaluated based on parameters such as: sum of squares of all errors (SStotal), sum of squares of standard errors (SSE), sum of squares of standard deviation (SSres), sum of squares of goodness of fit (SSlof), mean squared standard error (MSE).^20,22,24 In which, the square of the standard deviation was calculated according to formula and the variance values were calculated. The errors for lower values represent a higher degree of agreement between the predicted and actual values:

d_{i}^{2} = (y_{i j} - \bar{y})^{2}

(3)

where d_i is the standard deviation value, m is the number of levels of the model, n is the number of experimental runs, p is the number of parameters of the model,

\hat{y_{i}}

the predicted value of the model at this level level i,

\bar{y}

is the total mean value, y_ij is the repeatability value at each individual level,

\bar{y_{i}}

is the mean value of the central experiment.

Evaluate Antibacterial Ability

Prepare the culture medium: LB-Miller agar medium (g.L⁻¹) consists of components peptone 10 g, yeast extract 5 g, NaCl 5 g, agar 20 g, water 1 liter. LB-Miller medium (g.L⁻¹) consists of components peptone 10 g, yeast extract 5 g, NaCl 5 g, water 1 L.

Agar disk-diffusion method: Bacteria were cultured in liquid LB medium, diluted to a density of about 10⁶ CFU.mL⁻¹, and spread on LB agar medium.^25,26 Four wells (6 mm diameter) were punched on the surface of the agar plate and added 10 µL of extracts at different concentrations. 10 µL of amoxicillin antibiotic (concentration at 40 µg.mL⁻¹) and distilled water were used as positive and negative controls. The samples were then incubated at 37 °C for 24 h. The diameter of the inhibition zone (zone of resistance) was measured in millimeter (mm). The experiment was carried out on gram-negative (E. coli) and gram-positive (S. aureus) bacteria strains.

The minimum inhibitory concentration (MIC) determination: The bacterial culture was diluted to a density of approximately 10⁶ CFU.mL⁻¹. The extract was diluted to concentrations ranging from 0 mg.mL⁻¹ to 30 mg.mL⁻¹. The sample was inoculated in small volumes using a 96-well microtitration plate (microdilution).^25,26 Each well was loaded with 100 µL of sample and 100 µL of bacterial suspension. The controls consisted of water-containing samples and antibiotic-containing samples. 10 µL of Resazurin powder (concentration of 1 mg.mL⁻¹) was added to all wells. MIC was determined by observing the lowest concentration of extract that exhibits a color change (capable of inhibiting bacterial growth).^27-30

Results and Discussion

Evaluation of Raw KP Powder

KP was purchased from gardens in the Seven Mountains Region, An Giang Province, and identified at the Ginseng and Medicinal Center of Ho Chi Minh City. The results of the plant variety's morphological analysis (Figure 1) showed characteristics consistent with the plant species with the scientific name Kaempferia parviflora Wall.ex Baker, belonging to the genus Kaempferia, family Zingiberaceae. The plant, 28 cm long with an 18 mm thick tuber, has round leaves 24 cm long and many secondary roots.

Figure 1.

Sample Images of KP Plant in An Giang Province.

After drying and grinding, KP was obtained as a coarse powder because more than 95% of the particles passed through sieve No. 2000. The coarse powder was defined based on the powder size and sieve standards in the Vietnamese Pharmacopoeia V-Appendix 3.5-page PL119.³¹ The color value and basic quality parameters of KP powder were evaluated based on Vietnamese Pharmacopoeia V - Appendix 12.2 - page PL271³¹ and were presented in Table 2. The color of KP powder was evaluated through the L* (43.28), a* (8.1), and b* (6.83) values. Based on the color space (Commission Internationale de l'éclairage Laboratory Color Space – CIELAB color space), the powder sample was identified as Wenge color. The KP powder had a moisture content of 2.41 ± 0.23%, indicating that the powder was completely dry with a low moisture content of <10%. In addition, the values of total ash, soluble ash and insoluble ash were also calculated with values of 5.17%, 2.56% and 1.09% respectively. This shows that the content of organic compounds was quite high.

Table 2.

Quality Criteria of KP Powder.

Parameter	Unit	Value	Sensory
Parameter	Unit	Value	Odor	Taste
Color	L*	84.4	KP powder had a mild aroma typical of ginger and turmeric	KP powder had a slightly bitter taste on the tip of the tongue
	a*	0.96
	b*	17.03
Humidity	%	2.41 ± 0.23
Loss on Drying	%	64.36 ± 1.52
Total ash	%	5.17 ± 0.002
water-insoluble ash	%	2.56 ± 0.002
water-soluble ash	%	1.09 ± 0.004

Evaluate Factors Affecting the Extraction Process

Polyphenol and flavonoid content were greatly affected by extraction conditions. Therefore, influencing factors such as time, solvent ratio and extraction temperature were evaluated and repeated three times. The effect of extraction time was shown in Figure 2 with point at 30 min, 60 min, and 90 min. The results showed that polyphenol and flavonoid content tended to increase with increasing time from 60 min to 90 min. Therefore, 90 min was chosen as the time to evaluate the next factors.

Figure 2.

Effect of Extraction Time (A), Solvent Ratio (B), and Extraction Temperature (C).

The effect of solvent was shown in Figure 2. The results showed that polyphenol and flavonoid content was obtained at volume values of 5 mL, 10 mL, and 15 mL. The polyphenol content was increased with the increasing solvent volume while the flavonoid content was decreased. This showed that many organic compounds in the polyphenol and flavonoid groups were extracted, but only about 10–25 mg.(gDW)⁻¹ (accounting for about 40-60%). It can be seen that at the ratio of 1:15 g.mL⁻¹, the highest content of polyphenols and flavonoids were extracted. Therefore, the ratio of 1:15 g.mL⁻¹ was chosen as the solvent to evaluate the next factors. The influence of extraction temperature was performed at the temperature ranges of 50 °C, 60 °C, and 70 °C as shown in Figure 2. The evaluation values were constructed using the central values selected at 90 min, 15 mL volume and 60 °C temperature.

Optimization of Extraction Conditions for KP Extract

From the best adsorption conditions, the RSM model was constructed based on the central values and boundary values. Based on the BBD, a total of 17 experiments were carried out. From the RSM model, the results recorded include the quadratic equation of the model, line graph, 3D plot between factors and ANOVA table. The quadratic equation describing the correlation between the response factor (y) and the independent variables was defined as follows:

Polyphenol ((mgGAE).(gDW)⁻¹) = 78,51 + 11,88*A + 0,34*B + 7,63*C – 0,79*AB – 10,25*AC + 5,40*BC – 24,10*A² – 22,88*B² – 1,15*C²

Favonoid ((mgQE).(gDW)⁻¹) = 48,918 + 6,39*A + 2,89*B + 7,40*C – 0,93*AB – 3,66*AC – 0,04*BC – 11,13*A² – 12,82*B² – 11,33*C²

Data for ANOVA and ANOVA for the regression equations were presented in Tables 3 and 4. The significance of regression models was determined by the correlation coefficient (R²), ρ-value and F value. In general, the smaller the ρ-value and the larger the F value, the more statistical significance was demonstrated model. The model was effective when ρ < 0.05, and ineffective when ρ > 0.05. A p-value less than 0.05 demonstrates the statistical significance of the effective factors (95% confidence level). The results of ANOVA analysis show that the model was statistically significant (ρ < 0.05), and the regression coefficient of the model R² = 0.999 and 0.993 > 0.75. That proved the model to be compatible with the experiment. The standard error of the model was <10% of the total error. that indicated the model to be compatible with the experiment. In particular, the Lack of fit value of the model was Polyphenol = 1.29; Flavonoid = 0.42 shows that the misfit was very small and insignificant compared to the standard error of the model.

Table 3.

Variance Analysis of Mathematical Models.

Parameter	Polyphenol	Flavonoid
SSres	5.09	19.49
SSlof	2.51	4.69
SSE	2.58	14.80
SStot	7228.29	2774.54
MSE	1.29	0.423
R²	0.9993	0.9930
Correction R²	0.9984	0.9839
Predicted R²	0.9939	0.9646

Table 4.

ANOVA Value of RSM Model.

Sample	Parameter	SSE	MSE	F-value	ρ -value
Polyphenol	Model	7223.20	802.58	1103.45	< 0.0001
	A-time	1232.67	1232.67	1694.78	< 0.0001
	B-volume	0.3057	0.3057	0.42	0.5374
	C-temperature	464.87	464.87	639.14	< 0.0001
	AB	1.69	1.69	2.32	0.1714
	AC	420.78	420.78	578.53	< 0.0001
	BC	107.46	107.46	147.74	< 0.0001
	A²	2437.87	2437.87	3351.78	< 0.0001
	B²	2236.75	2236.75	3075.26	< 0.0001
	C²	5.94	5.94	8.17	0.0244
Flavanoid	Model	2755.05	306.12	109.92	< 0.0001
	A-time	244.76	244.76	87.89	< 0.0001
	B-volume	25.67	25.67	9.22	0.0189
	C-temperature	466.80	466.80	167.62	< 0.0001
	AB	3.45	3.45	1.24	0.3027
	AC	54.25	54.25	19.48	0.0031
	BC	0.0056	0.0056	0.002	0.9655
	A²	522.55	522.55	187.64	< 0.0001
	B²	690.49	690.49	247.94	< 0.0001
	C²	541.78	541.78	194.54	< 0.0001

From the evaluation results, the line graph and 3D plot between factors were shown in Figure 3 and Figure 4, which show the highest area of the model for the optimum value of the material. The polyphenol content has a convergence point at the volume of 12 to 18 mL, the time of 75 to 115 min and the temperature of 50 to 150 °C. The surface graph and 3D model were shown the optimum area of the evaluation model. The interaction between the elements was shown the convergence in the 3D figure. For the Flavonoid content, there was a convergence point at the volume of 12 to 18 mL, the time of 75 to 115 min and the temperature of 50 to 80 °C. The surface graph and 3D model were shown the optimum area of the evaluation model. The interaction between the factors was shown the convergence in Figure 3D.

Figure 3.

Line Plot and Surface Plot of Polyphenol Content.

Figure 4.

Line Plot and Surface Plot of Flavonoid Content.

The optimal values were calculated and selected based on the optimal area from the influencing factors and shown in Figure 5. The conditions recorded at 96 min, 15.4 mL volume, and 66 °C temperature gave the polyphenol and flavonoid contents predicted by the model as 81.57 (mgGAE).(gDW)⁻¹ and 50.70 mgQE).(gDW)⁻¹ with the model's predicted regression coefficient of 99.6%.

Figure 5.

Optimal Value from RSM Model.

Evaluation of Antibacterial Ability

The antibacterial ability was performed based on qualitative (disk diffusion method) and quantitative (Minimum inhibitory concentration, MIC) methods and shown in Figure 6 and Table 5. The extract was mixed into different concentrations from 2 to 8 mg.mL⁻¹. For E. coli strain, the antibacterial zone at 8 mg.mL⁻¹ concentration had the largest antibacterial zone diameter of 16 mm and gradually decreased at lower concentrations. It can be seen that the resistance zone with a diameter of 6 mm was determined to have no antibacterial zone at a concentration of 2 mg.mL⁻¹. The antibacterial zone diameter for S. aureus strain, the antibacterial zone at 8 mg.mL⁻¹ concentration had an antibacterial zone diameter of 8 mm.

Figure 6.

Antibacterial Cycle of E. coli (A) and S. aureus (B) at Different Concentrations.

Table 5.

Evaluation of the Minimum Inhibitory Concentration of E.coli and S. aureus bacteria of KP Extract.

The MIC was evaluated and shown in Table 5. The sample was evaluated on two bacterial strains: E.coli (gram-negative) and S. aureus (gram-positive). Evaluation samples included positive control (antibiotic Chloramphenicol), negative control (bacteria), and extract (30 mg.mL⁻¹-2.5 mg.mL⁻¹). The results showed that the positive control sample was blue and the negative control sample was dark pink. The samples were evaluated for a change in color from purple to pink as the extract concentration gradually decreased. For E.coli strains, the solution was dark purple, gradually fading when it reached a concentration of 10 mg.mL⁻¹. The solution turned pink at a concentration of 2.5 mg.mL⁻¹ equivalent to the negative control sample, showing no ability to inhibit bacteria. This showed that concentrations of 5 and 7.5 mg.mL⁻¹ still partially inhibited bacteria in the sample. For S. aureus strains The solution was dark purple, gradually fading when it reached a concentration of 15 mg.mL⁻¹. The solution turned pink at a concentration of 2.5 mg.mL⁻¹ equivalent to the negative control sample, showing no ability to inhibit bacteria. This showed that concentrations of 10, 7.5 and 5 mg.mL⁻¹ still partially inhibited bacteria in the sample.

Evaluation of Antioxidant Capacity

The antioxidant effect of the KP extract was determined based on the DPPH free radical neutralization efficiency. The antioxidant results were shown in Table 6. KP extract gave an IC₅₀ value of ABTS of 0.015 ± 0.006 mg.mL⁻¹ and DPPH of 6.907 ± 1.014 mg.mL⁻¹.

Table 6.

IC₅₀ Values of DPPH and ABTS Free Radical Scavenging Methods.

Sample	IC₅₀ (mg.mL⁻¹)
Sample	ABTS	DPPH
Vitamin C	0.0113 ± 4.40 × 10⁻⁵	0.0023 ± 2.032 × 10⁻⁶
KP extract	0.015 ± 0.006	6.907 ± 1.014

Discussion

The plant species with the scientific name Kaempferia parviflora Wall.ex Baker, belonging to the genus Kaempferia, family Zingiberaceae. The results showed that polyphenol and flavonoid content tended to increase with increasing time from 60 min to 90 min. In Zuraida Ab Rahman's study, similar results were found when evaluating the influence of time on the content of polyphenols and flavonoids. The content of polyphenols and flavonoids were tended to increase with increasing extraction time.¹⁵

The results showed that polyphenol content tends to increase with increasing extraction temperature, while flavonoid content decreases with increasing extraction temperature. In the study of Rahman and his colleagues, it was shown that the polyphenol content increased as the temperature increased, but the flavonoid content didn’t change much when the extraction temperature increased.¹⁵ This shown that temperature had a great influence on the ability to release organic compounds in KP powder. However, the polyphenol and flavonoid contents didn’t change significantly. Therefore, an optimization model was performed to evaluate the process comprehensively.

Therefore, the experiment was repeated 3 times with optimal conditions. The results were recorded with the first experiment: polyphenol = 78,422 (mgGAE).(gDW)⁻¹, flavonoid = 51,532 (mgQE).(gDW)⁻¹; second experiment: polyphenol = 82,683 (mgGAE).(gDW)⁻¹, flavonoid = 48,902 (mgQE).(gDW)⁻¹, third experiment: polyphenol = 81,043 (mgGAE).(gDW)⁻¹, flavonoid = 49,925 (mgQE).(gDW)⁻¹. The experimental results show that the predicted model is very compatible with the experiment.

The sample was evaluated on two bacterial strains: E.coli (gram-negative) and S. aureus (gram-positive). Lower concentrations were recorded as having no antibacterial zone. The results of MIC evaluation showed that KP extract had better resistance to gram-negative bacteria than gram-positive bacteria when the MIC of E.coli was 10 mg.mL⁻¹ and S. aureus was 15 mg.mL⁻¹.

It can be seen that the MIC of 50% of the extract from the DPPH method had a higher concentration value than that of the ABTS method, corresponding to lower activity. This was explained in Melanie Platzer's study on the interaction of organic compounds with the DPPH and ABTS methods. Compounds into the flavanone group should interact more easily with ABTS than DPPH through the sequential proton loss electron transfer (SPLET) method.³² It could be seen that the organic compounds present in KP to be mostly flavanone group.

Conclusion

Kaempferia parviflora was purchased from gardens in the Seven Mountains Region – An Giang Province and identified as Kaempferia parviflora Wall.ex Baker species belonging to the genus Kaempferia, family Zingiberaceae. KP was dried into raw powder and evaluated based on Vietnam Pharmacopoeia V – appendix 12.2 – page PL271. The results showed that the raw powder had a dark purple color (Wenge), a light aroma (characteristic of ginger and turmeric), and a bitter taste. The moisture, total ash, soluble ash, and insoluble ash of the powder were recorded with values of 2.41%, 5.17%, 2.56%, and 1.09%. The results of the study on the influence of extraction conditions showed that temperature and time factors had a certain influence on the polyphenol and flavonoid content. It could be seen that with a time of 90 min, a volume of 15 mL and a temperature from 50 to 70 °C, polyphenol and flavonoid contents were obtained 55 (mgGAE).(gDW)⁻¹ and 45 (mgQE).(gDW)⁻¹, respectively. The polyphenol and flavonoid contents were evaluated based on the RSM using the Box-benken model with a total of 17 experiments and the central value repeated 5 times. The model parameters had shown agreement between the experimental value and the predicted value from the model with the model's regression coefficient R² = 0.999 and 0.993 > 0.75. In optimal conditions (at 96 min, volume 15.4 mL, temperature 66 °C), polyphenol and flavonoid contents were predicted from the model to be 81.57 (mgGAE).(gDW)⁻¹ and 50.70 (mgQE).(gDW)⁻¹. Besides, the antibacterial ability of the sample was also recorded at the MIC of Escherichia coli of 10 mg.mL⁻¹ and Staphylococcus aureus of 15 mg.mL⁻¹. The high antioxidant capacity was found with DPPH and ABTS to be 6.990 and 0.014 mg.mL⁻¹. An evaluation of the extraction process parameters was conducted to optimize the production of KP on an industrial scale. The study highlights that KP extract has significant potential for applications in medicine and cosmetics. However, the study's limitations included not evaluating the activities provided by KP, a small sample size, and the absence of in vivo studies. This forms the basis for further in-depth research as well as practical applications of KP.

Footnotes

ORCID iDs

Thien Hien Tran

Bich Ngoc Hoang

Ethical Approval

Ethical Approval is not applicable for this article.

Author Contributions

Writing-original draft preparation B.N.H and T.T.H.; data curation T.T.N and T.T.T.; methodology L.G.B and T.T.H; writing-review and editing B.N.H. and L.G.B. All authors have assented to the manuscript's published version.

Funding

This research is funded by Nguyen Tat Thanh University, Ho Chi Minh City, Vietnam under grant number SPUD.2024.01.16.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability

Data included in article/Supplemental material/referenced in article.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

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