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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 36  |  Issue : 2  |  Page : 69-77

Phytochemical analysis and potential applications of the ethanol and aqueous-ethanol extracts of some selected plant in family Zingiberaceae plants for cosmeceutical and health-promoting food


1 Department of Medical Sciences, Faculty of Science, Rangsit University, Pathum Thani, Thailand
2 Faculty of Medical Technology, Rangsit University, Pathum Thani, Thailand
3 Sun Herb, Thai, Chinese Manufacturing, Rangsit University, Pathum Thani, Thailand
4 Drug and Herbal Product Research and Development Center, College of Pharmacy, Rangsit University, Pathum Thani, Thailand

Date of Submission17-Dec-2021
Date of Decision02-Jan-2022
Date of Acceptance21-Feb-2022
Date of Web Publication16-Nov-2022

Correspondence Address:
Dr. Pannapa Powthong
Department of Medical Sciences, Faculty of Science, Rangsit University, Mueang Pathum Thani 12000
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jms.jms_145_21

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  Abstract 


Background: The rhizomes of the Zingiberaceae family are a vegetable widely used in many Asian nations, and their therapeutic properties have been acknowledged in many traditional recipes.
Aims and Objectives: Investigate the in vitro biological effect of the aqueous-ethanol and ethanol crude extract received from three medicinal plants in the family Zingiberaceae.
Materials and Methods: Three species of Zingiberaceae plants including Curcuma longa L., Curcuma zedoaria (Christm.) and Curcuma aromatica Salisb.were gathered and evaluated for their phytochemical contents, anti-inflammatory and anti-oxidant characteristics using the aqueous-ethanol (30:70%) and ethanol (95%) extraction and varying according to single and mixed extracts (1:1:1 and 2:1:1 ratio respectively) for determining the synergistic effects.
Results: It was indicated that extracts of the three selected plant contained at least 5 from 13 phytochemical constituents. The single aqueous-ethanol extract of C. aromatica Salisb. and synergy achieved at 1:1:1 ratio of aqueous-ethanol extract showed the highest effective anti-inflammatory activity. The greatest antioxidant activity was found in a single ethanol extract of C. zedoaria (Christm.) and synergistically obtained at a 1:1:1 ratio of aqueous-ethanol extract. Furthermore, we discovered that combination extract produced greater outcomes than utilizing the mono extract alone.
Conclusion: Our results demonstrate that screening for chosen Zingiberaceae plant extracts is a favorable representation of the value of screening for cosmetically and medicinal purposes.

Keywords: Curcuma aromatica, Curcuma longa, Curcuma zedoaria, Zingiberaceae


How to cite this article:
Powthong P, Lektrakul W, Juntrapanukorn B, Luprasong C, Monton C. Phytochemical analysis and potential applications of the ethanol and aqueous-ethanol extracts of some selected plant in family Zingiberaceae plants for cosmeceutical and health-promoting food. J Med Soc 2022;36:69-77

How to cite this URL:
Powthong P, Lektrakul W, Juntrapanukorn B, Luprasong C, Monton C. Phytochemical analysis and potential applications of the ethanol and aqueous-ethanol extracts of some selected plant in family Zingiberaceae plants for cosmeceutical and health-promoting food. J Med Soc [serial online] 2022 [cited 2022 Nov 28];36:69-77. Available from: https://www.jmedsoc.org/text.asp?2022/36/2/69/361281




  Introduction Top


Family Zingiberaceae has around 85 species of plants that are mostly grown in Asia, Central and South America, and Africa.[1] They are recognized by their tuberous or nontuberous rhizomes, which contain aromatic and therapeutic uses. Almost plants have been used by humankind as a source of food (spices and flavoring agents) and traditional medicine. Their rhizomes possess a number of medicinal, pharmacological, and nutritional properties such as anti-inflammatory, immunostimulatory, immunomodulatory, and anticancer activity.[2],[3] Although members of this genus have a similar appearance, their pharmacological and therapeutic activities range greatly.[4]

Environmental stress can cause overproduction of oxidative stress, which is defined by cell/tissue injury and oxidative macromolecule damage. Oxidative stress can cause a number of diseases and accelerate aging, including atherosclerosis, diabetes, and cancer.[5] Even though the human body is created to have its own defense and repair systems to defend against oxidative damage, these systems are insufficient to completely avoid injury. Many reports have been indicating that many Zingiberaceae plants contain antioxidants and they may be useful in reducing oxidative damage.[6] The therapeutic effect and dose intake of natural extracts, on the other hand, vary owing to changes in the extraction method and active components included in it.

Our objective was to investigate the in vitro biological effect of the aqueous-ethanol and ethanol crude extracts received from three medicinal plants in the family Zingiberaceae including turmeric (Curcuma longa L.), zedoary (Curcuma zedoaria (Christm.), and wild turmeric (Curcuma aromatica Salisb.) on phytochemical constituent, anti-inflammatory, and antioxidant activity. The scientific result obtained here was the possibility of developing this variety as an ingredient in health/medical cosmeceutical purposes or an alternative treatment to reduce anti-inflammatory drug side effects.


  Materials and Methods Top


Plant material

Three medicinal plants in the family Zingiberaceae, including turmeric, zedoary, and wild turmeric, were purchased from the local market in Pathum Thani, Thailand. A taxonomist from the Drug and Herbal Product Research and Development Center, College of Pharmacy, Rangsit University, validated the botanical identity of each plant specimen. It was authenticated to be C. longa L., C. zedoaria (Christm.), and Curcuma aromatica Salisb. belonging to the family Zingiberaceae. Following that, the plant was washed, cut into little pieces, and dried at 40°C for 15–20 h. The dried material was crushed into powder and kept in an airtight plastic bag at room temperature in a desiccator for subsequent examination.

Chemicals and reagents

Bovine serum albumin, butylated hydroxyl toluene, trypsin, Tris-HCl, thiobarbituric acid (TBA), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich Co., St. Louis, USA. All other basic reagents were of analytical grade.

Preparation of ethanolic crude extract

Ethanolic extraction was carried out for 5 days at 37°C using a sample of plant powders: 95% ethanol equivalent to a 1:10 ratio. The solid has been removed by filtering it using Whatman No. 1 filter paper. For ethanol removal, the filtrate was processed through a rotary evaporator. Following that, each plant ethanolic crude extract was obtained. The crude was redissolved in DMSO at a concentration of 1 g/mL and stored in a glass container at −20°C for subsequent analysis during the experiment.

The same technique was followed for aqueous-ethanol extraction with 30% distilled water: 70% ethanol.

Preliminary phytochemical screening of plant crude extract

Each plant crude extract was subjected to the following assays to assess the presence or absence of fundamental phytochemicals: anthraquinones, tannins, saponins, flavonoids, glycosides, cardiac glycosides, terpenoids, steroids, alkaloids, coumarins, phenols, protein and amino acids, and fat and oil using standard biochemical procedures.[7],[8]

Anti-inflammatory activity of plant extract

Inhibition of albumin denaturation

The anti-inflammatory activity of each plant in the family Zingiberaceae was studied by using the inhibition of albumin denaturation technique which was studied according to previous studies,[9],[10] followed by minor modifications. The reaction mixture consisted of 100 μl of test extract samples at different concentrations (50–5000 μg/mL) and 500 μl of 1% aqueous solution of bovine albumin fraction. The sample extracts were incubated at 37°C for 20 min and then heated to 51°C for 20 min; after cooling the samples, the turbidity was measured at 660 nm. The positive and negative control was acetylsalicylic acid and distilled water, respectively. The experiment was performed in triplicate. The percentage inhibition of protein denaturation was calculated as follows:



where A0 is the absorbance of the control (control contained 1% aqueous solution of bovine albumin and distilled water), and A1 is the absorbance of the sample.

Inhibition of protein denaturation method using egg albumin

The anti-inflammatory effect of each plant in the Zingiberaceae family was investigated using the inhibition of egg albumin denaturation approach described before,[11],[12] with minor changes. The reaction mixture contained 200 μl of test extracts at various concentrations (50–5000 μg/mL), 20 μl of eggs albumin (from hen egg), and 280 μl of phosphate-buffered saline (pH 6.4). The sample extracts were incubated at 37°C for 15 min and then heated to 70°C for 15 min; after cooling the samples, the turbidity was measured at 660 nm. Acetylsalicylic acid and distilled water were used as positive and negative controls, respectively. The experiment was carried out three times. The following percentage inhibition of protein denaturation was calculated:



where A0 is the absorbance of the negative control, and A1 is the absorbance of the sample.

In vitro proteinase inhibitory assay

The in vitro proteinase inhibitory was performed as described previously.[13] Briefly, the test extract at various concentrations (50–5000 μg/mL) was prepared. Then, 500 of each concentration was mixed with 500 μL of 0.06 mg of trypsin in 20 mM Tris-HCl buffer (pH 7.4). The mixture was incubated at 37°C for 5 min, and 500 μL of 0.8% (W/V) casein was added. The mixture was re-incubated at 37°C for another 20 min, and then, 1000 μL of 10% glacial acetic acid was added to stop the reaction. The mixture tube was then centrifuged at 3000 rpm for 10 min. The resulting supernatant was subjected to measure the absorbance at a wavelength of 210 nm. The experiment was performed triplicated. The following percentage of inhibition proteinase inhibitory activity was calculated:



where A0 is the absorbance of the negative control (distilled water), and A1 is the absorbance of the sample.

Antioxidant assay of plant extract

Lipid peroxidase

Malondialdehyde, the last product of lipid oxidation, was measured with TBA reactive substance method as modified from a previous study.[14] Briefly, 50-5000 μg/mL of plant extract dilution was prepared with water as a solvent. 50 μl of this solution was mixed with 50 μl of egg yolk 10% w/v KCl solution, 300 μl of 20% w/v acetic acid (pH 3.5), and 300 μl of TBA solution. The mixture then was boiled at 95°C for 1 h. Then, 750 μl of butanol was added and leave it until its color as changed into pink. The mixture was then centrifuged at 3000 g at 25°C for 10 min. Supernatant absorbance was measured at 532 nm. Alpha-tocopherol and distilled water were used as positive and negative controls, respectively. The experiment was carried out three times. Lipid peroxidation inhibition percentage was calculated by the following formula:



where A0 is the absorbance of the negative control (distilled water), and A1 is the absorbance of the sample.

Metal-chelating complex assay

The ferrous ion-chelating activity of methanol and aqueous-ethanol extracts was estimated by the method of Le et al.[15] Briefly, 100 μL of the extract samples at different concentrations (50–5000 μg/mL) was added to a solution of 2 mM FeCl2 (10 μL). The reaction was initiated by the addition of 20 μL of ferrozine (5 mM), and the mixture was shaken vigorously and left standing at room temperature for 10 min. The absorbance of the solution was then measured at 562 nm. Ascorbic acid was used as a positive control. The experiment was performed in triplicate. The percentage of inhibition of ferrozine −Fe2+ complex formation was calculated using the following formula:



where A0 is the absorbance of the control (control contained FeCl2 and ferrozine; complex formation molecules), and A1 is the absorbance of the sample.

Hydrogen peroxide scavenging activity

In the present study, the ability of plant extracts to scavenge hydrogen peroxide can be modified according to the previous report.[16] Briefly, 200 μl of various concentrations of plant extract concentration (50–5000 μg/mL) in methanol was mixed with 100 μl ml of a solution of hydrogen peroxide (20 mM) in phosphate buffer (50 mM pH 7.4). The mixture was left to stand for 10 min, and the absorbance was then measured at 230 nm against a blank solution containing phosphate buffer without hydrogen peroxide. The assay was carried out in triplicate. Alpha-tocopherol was used as a positive control. The percentage of hydrogen peroxide scavenging is calculated as follows:

% Scavenged (H2O2) = ([Ai − At]/Ai) ×100

where Ai is the absorbance of the control, and At is the absorbance of the test.

Statistical analysis

All the experiments were performed and studied in triplicate. Results represent the mean ± standard deviation. Student's t-test was performed to test the significance of differences between means at the 5% level using the SPSS ver. 22 software (Chicago, IL, USA) to conduct the statistical analysis.


  Results Top


Phytochemical analysis

As shown in [Table 1], the aqueous-ethanol and ethanol crude extracts received from three medicinal plants in the family Zingiberaceae were found to possess various phytochemicals or polyphenols such as flavonoids, phenolics, steroids, glycosides, saponins, coumarins, protein/amino acids, and fat/oil. Furthermore, it was discovered that each solvent allowed the identification of the same phytochemical except for protein and amino acids and saponins in the aqueous-ethanol crude extract of C. longa L and C. zedoaria (Christm.), but not in ethanol solvent.
Table 1: Phytochemical result of plant crude extract

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Inhibition of albumin denaturation

Protein denaturation is a well-known cause of inflammation. The capacity of the plant extract to prevent protein denaturation was investigated as part of the study into the mechanism of anti-inflammation action. It was denoted that heat-induced albumin denaturation was inhibited by Zingiberaceae plants [Figure 1] and [Table 2]. Within a concentration range of 50–5000 g/mL, the percentage of inhibition was measured to be 30.27%–93.42%. Maximum inhibition was observed from aqueous-ethanol extract of C. aromatica Salisb. with an IC50 value of 0.61 μg/mL, followed by ethanol extract of C. zedoaria (Christm.) and aqueous-ethanol extract of C. zedoaria (Christm.) (IC50 value: 2.79 and 4.81 μg/ml, respectively). The 1:1:1 ratio of aqueous-ethanol demonstrated the strongest activity (IC50 value: 3.50 μg/ml). The t-test analysis showed that there is a significant difference in the level of inhibition compared to acetylsalicylic acid, a standard anti-inflammation drug which showed a maximum inhibition of 93.03% at the concentration of 5000 μg/mL (IC50 value: 26.88 μg/mL).
Figure 1: Anti-inflammatory assays of different concentrations of aqueous-ethanol and ethanol extracts of C. longa L., C. zedoaria (Christm.), and Curcuma aromatica Salisb. (a) The inhibition of protein denaturation of aqueous-ethanol extract determined in various concentrations varying from 50 to 5000 μg/mL; (a') The inhibition of protein denaturation of ethanol extract was evaluated in concentration varying from 50 to 5000 μg/mL. A gradual increase in scavenging potential of the extract was obtained with an increase in concentration; (b) The inhibition of protein denaturation method using egg albumin of aqueous-ethanol extract; (b') The inhibition of protein denaturation method using egg albumin of ethanol extract. A gradual increase in scavenging potential of the extract was obtained with an increase in concentration; (c) The inhibition of proteinase activity of aqueous-ethanol extract was determined; (c') The inhibition of proteinase activity of ethanol extract was determined. A gradual increase in scavenging potential of the extract was obtained with an increase in concentration; n = 3 (mean ± standard deviation)

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Table 2: IC50 values for anti-inflammation activities by plant extracts and standard

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Inhibition of egg albumin denaturation

The anti-inflammatory properties of Zingiberaceae plants were tested in vitro against the denaturation of egg albumin. The present findings exhibited a concentration-dependent inhibition of egg protein (albumin) denaturation by most of the extracts. The percentage of inhibition was found to be 4.29%–81.61%. Aqueous-ethanol extract C. aromatica Salisb. has the maximum level of albumin denaturation inhibition with an IC50 value of 810.12 μg/mL, followed by aqueous-ethanol extract of C. zedoaria (Christm.) and ethanol extract of C. aromatica Salisb. (IC50 value: 842.44 and 971.90 μg/ml, respectively). The 2:1:1 ratio of aqueous-ethanol demonstrated the strongest activity (IC50 value: 9.01 μg/ml). The t-test analysis showed that there is a significant difference in the level of inhibition compared with the standard (acetylsalicylic acid) and it was 65.24%–92.20% (IC50 value: 180.15 μg/ml) throughout the concentration range of 50–5000 μg/ml, as shown in [Figure 1] and [Table 2].
Figure 2: Antioxidant assays of different concentrations of aqueous-ethanol and ethanol extracts of C. longa L., C. zedoaria (Christm.), and Curcuma aromatica Salisb. (a) Anti-lipid peroxidase activity of aqueous-ethanol extract determined in various concentrations varying from 50 to 5000 μg/mL; (a') Anti-lipid peroxidase activity of ethanol extract was evaluated in concentration varying from 50 to 5000 μg/mL. A gradual increase in scavenging potential of the extract was obtained with an increase in concentration; (b) Metal-chelating complex activity of aqueous-ethanol extract; (b') Metal-chelating complex activity of ethanol extract. A gradual increase in scavenging potential of the extract was obtained with an increase in concentration. (c) Hydrogen peroxide scavenging activity of aqueous-ethanol extract was determined; (c') Hydrogen peroxide scavenging activity of ethanol extract was determined. A gradual increase in scavenging potential of the extract was obtained with an increase in concentration; n = 3 (mean ± standard deviation)

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Proteinase inhibitory activity

Proteinases have been linked to arthritic symptoms. Neutrophil lysosomal granules are known to be a rich source of proteinase. All extracts from the Zingiberaceae family demonstrated substantial anti-proteinase activity in a concentration-dependent manner. The majority of them showed more than 90% inhibition at the lowest concentration of 50 μg/ml. The percentage of inhibition was found to be 90.18%–100%. The highest value of proteinase inhibitory activity was detected in ethanol extract of C. aromatica Salisb. with an IC50 value of 398.92 μg/mL, followed by aqueous-ethanol extract of C. aromatica Salisb. and aqueous-ethanol extract of C. zedoaria (Christm.) with an IC50 value of 400.97 and 401.96 μg/mL, respectively. The 1:1:1 ratio of aqueous-ethanol demonstrated the strongest activity (IC50 value: 399.66 μg/ml), as shown in [Figure 1] and [Table 2].

Anti-lipid peroxidation with reactive substance method

From [Figure 2] and [Table 3], it showed that all extracts of the fascinating plants in the Zingiberaceae family were capable to inhibit egg yolk cholesterol peroxidation reaction in a concentration-dependent manner compared to the blank. The inhibition percentage was determined to be 6.45%–54.81%. The highest value of anti-lipid peroxidation was denoted in ethanol extract of C. zedoaria (Christm.) with an IC50 value of 3.64 μg/mL, follow by aqueous-ethanol extract of C. zedoaria (Christm.) and ethanol extract of C. longa L. with an IC50 value of 10.54 and 119.60 μg/mL respectively. The 1:1:1 ratio of aqueous-ethanol demonstrated the strongest activity (IC50 value: 2.37 μg/ml). The t-test analysis revealed a significant difference in the amount of inhibition when compared to standard alpha-tocopherol (IC50 448.56 μg/mL), as shown in [Figure 2] and [Table 3].

Ferrous ion-chelating activity

All extracts of the interesting plants in the family Zingiberaceae were able to chelate ferrous ions in a concentration-dependent manner. Across the whole concentration range of 50–5000 g/ml, the inhibition percentage was estimated to be 2.56%–71.69%. At the lowest concentration of 400 μg/mL, aqueous-ethanol extract of C. longa L. exerted the strongest chelating effect (53.31%) with an IC50 value of 127.17 μg/mL, followed by ethanol extract of C. aromatica Salisb. and aqueous-ethanol extract of C. aromatica Salisb. (IC50 value of 422.89 and 685.34 μg/ml, respectively). The 1:1:1 ratio of aqueous-ethanol revealed the highest activity (IC50 value: 504.73 μg/ml). This activity was similar to that obtained with the standard chelator ascorbic acid (IC50: 448.56 μg/mL), as shown in [Figure 2] and [Table 3].
Table 3: IC50 values for hydrogen peroxide scavenging activity by plant extracts and standard

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Hydrogen peroxide scavenging activity

The aqueous-ethanol and ethanol crude extracts were screened for H2O2 radical scavenging activity, in which the level of inhibition was calculated to be 17.52%–100%. The highest activity was detected in ethanol extract of C. zedoaria (Christm.) at the lowest concentration of 50 μg/mL with an IC50 value of 5.09 μg/ml, followed by aqueous-ethanol extract of C. aromatica Salisb. and C. longa L. (IC50 value: 6.93 and 19.13 μg/ml, respectively). The 2:1:1 ratio of aqueous-ethanol showed the highest activity (IC50 value: 7.33 μg/ml). The t-test analysis showed that there is a significant difference in the H2O2 radical scavenging activity among the different extracts of the test sample and standard alpha-tocopherol with an IC 50 value of 398.74 μg/ml, as shown in [Figure 2] and [Table 3].


  Discussion Top


The results of preliminary quantitative phytochemical screening of aqueous-ethanol and ethanol extracts revealed the presence of multiple polar and nonpolar chemical constituents. Flavonoids, phenolics, steroids, glycosides, saponins, coumarins, protein/amino acids, and fat/oil were present in the extracts. Aqueous-ethanol extraction allowed some phytochemicals present than in ethanol extraction. It was correlated with a previous study report that many essential oils are found in Zingiberaceae plants, including terpenes, alcohols, ketones, flavonoids, carotenoids, and phytoestrogens which can be extracted using organic solvents.[17],[18],[19],[20] Curcuminoids, which include curcumin, demethoxycurcumin, and bisdemethoxycurcumin, have been identified as the major active components.[21] Several studies have discovered that plant extracts high in polyphenols and important phytoconstituents such as alkaloids, glycosides, terpenoids, saponins, phenols, and steroids exhibit significant antioxidant and free radical scavenging activities in a variety of antioxidant models.[22],[23]

Although numerous procedures for assessing the antioxidant activity of biological samples have been developed, it is extremely difficult to analyze each antioxidant component independently since each antioxidant molecule is difficult to isolate from organic extracts. As a result, we aimed to confirm the biological efficacy in different in vitro antioxidant models by using crude extract in this study.

Protein denaturation and proteinases in arthritic responses have been implicated as a source of inflammation. The capacity of the aqueous-ethanol and ethanol extracts to prevent protein denaturation in both bovine serum and egg albumin was evaluated as part of the experiment on the mechanism of anti-inflammation action. Currently, the single aqueous-ethanol extract of C. aromatica Salisb. and synergy achieved at a 1:1:1 ratio of aqueous-ethanol extract exhibited the effectively prevented heat-induced protein denaturation and proteinase inhibition compared to acetylsalicylic acid, a standard anti-inflammation drug at the concentration of 50–5000 μg/ml.

Previous studies report that curcumin has anti-inflammatory effects,[24],[25] and one of its mechanisms is most likely mediated by its capacity to inhibit cyclooxygenase-2, lipoxygenase, and inducible nitric oxide synthase which are key enzymes that mediate inflammatory processes.[26],[27] Oral curcumin has been demonstrated in animal experiments to produce antinociceptive effects,[28] with the involvement of ATP-sensitive potassium channels.[29] Curcumin has shown potential in pilot human trials for relieving symptoms of rheumatoid arthritis and inflammatory bowel illness.[30]

Oxidative stress enhanced several diseases, including cancer, diabetes, metabolic disorders, atherosclerosis, and cardiovascular diseases. Antioxidant properties in the extract applied in this study demonstrated effective in vitro findings, including anti-lipid-peroxidation, ferrous ion-chelating activity, and hydrogen peroxide scavenging. The single ethanol extract of C. zedoaria (Christm.) and synergy obtained at a 1:1:1 aqueous-ethanol extract ratio effectively prevented harmful ROS. The antioxidant activity of plants is mostly acquired from active molecules or major phytoconstituents that they possess.[31] Indeed, studies have revealed that curcumin, the primary active component, may rapidly scavenge free radicals, mainly to its H-atom donation from the phenolic group. Furthermore, it is associated with the scavenging of these radicals in peroxidation processes and is a possible antioxidant against the production of hydrogen peroxide and superoxide radicals.[32],[33],[34]

Regarding the extraction methods used, the combination between water and ethanol and ethanol alone was used to obtain good and effective results. It was demonstrated that aqueous-ethanol extraction method and synergistic effects among the combinations of selected plants in this group on the anti-inflammation and antioxidant were documented.


  Conclusion Top


The above results conclude that the rhizome of a selected plant in the Zingiberaceae family contains polyherbal formulations with varying concentrations that have anti-inflammation and antioxidant properties valuable enough to warrant development as a safe cosmeceutical or functional food product for anti-aging and to reduce inflammatory disease. Furthermore, this study suggests that combining the selected plants in this group may be utilized as a novel method, allowing us to employ a lower concentration of the extract or active components, lowering the possibility of harmful consequences.

Acknowledgments

The first author would like to express her sincere gratitude and profound appreciation to Research Institute and also Faculty of Medical Technology, Rangsit University for providing funds to P. Powthong. The authors would like to extend their thanks to Sun Herb Thai Chinese Manufacturing, and also Drug and Herbal Product Research and Development Center, College of Pharmacy, Rangsit University for providing plant samples.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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