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ORIGINAL ARTICLE
Year : 2020  |  Volume : 11  |  Issue : 4  |  Page : 174-178  

Anti-aging potential and phytochemicals of Centella asiatica, Nelumbo nucifera, and Hibiscus sabdariffa extracts


1 Department of Health and Aesthetics, Thai Traditional Medicine College, Rajamangala University of Technology Thanyaburi, Pathum Thani, Thailand
2 Department of Innovation of Health Products, Thai Traditional Medicine College, Rajamangala University of Technology Thanyaburi, Pathum Thani, Thailand
3 Department of Applied Thai Traditional Medicine, Thai Traditional Medicine College, Rajamangala University of Technology Thanyaburi, Pathum Thani, Thailand

Date of Submission11-Jun-2020
Date of Decision16-Jul-2020
Date of Acceptance20-Jul-2020
Date of Web Publication10-Oct-2020

Correspondence Address:
Miss. Monsicha Khuanekkaphan
No. 8 Phahonyothin, 87 Prachathipat Thanyaburi, Pathum Thani 12130
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/japtr.JAPTR_79_20

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  Abstract 


Centella asiatica, Nelumbo nucifera Gaertn, and Hibiscus sabdariffa have been used as medicinal plants in Thailand. They are sources of phytochemicals that applications for esthetic and healthcare. The aim of this research was to examine the phytochemical constituents and anti-aging potential of these plants. The phytochemical compounds were performed using gas chromatography-mass spectrometry. The anti-aging activities were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis (3-ethylbenzothiazoline-6-sunfonic acid) (ABTS), anti-collagenase, and anti-elastase assays. The main interest phytochemical compounds of ethanolic extracts of C. asiatica, N. nucifera, H. sabdariffa were ethanol, 2-(-Octadecenyloxy), γ-sitosterol and hexadecanoic acid, and ethyl ester, respectively. The DPPH half-maximal inhibitory concentration (IC50) results of C. asiatica, N. nucifera, and H. sabdariffa were 0.32 ± 0.01, 0.34 ± 0.00, and 0.35 ± 0.01 mg/mL, respectively. The ABTS result of H. sabdariffa extract showed high inhibitory activity at IC50of the extract was 0.62 ± 0.12 mg/mL. The percentage of collagenase inhibition of C. asiatica, N. nucifera, and H. sabdariffa at 1.0 mg/mL was 78.13 ± 4.42, 85.94 ± 2.21, and 90.63 ± 0.00, respectively. The C. asiatica extract had a high percentage of elastase inhibition. Consequently, these research results suggest that phytochemicals may also provide a range of esthetic and health benefits. The phytochemical constituent could be used as anti-aging active ingredient for cosmetic and pharmaceutical industrials.

Keywords: Anti-aging, Centella asiatica, Hibiscus sabdariffa, Nelumbo nucifera, phytochemical


How to cite this article:
Khuanekkaphan M, Noysang C, Khobjai W. Anti-aging potential and phytochemicals of Centella asiatica, Nelumbo nucifera, and Hibiscus sabdariffa extracts. J Adv Pharm Technol Res 2020;11:174-8

How to cite this URL:
Khuanekkaphan M, Noysang C, Khobjai W. Anti-aging potential and phytochemicals of Centella asiatica, Nelumbo nucifera, and Hibiscus sabdariffa extracts. J Adv Pharm Technol Res [serial online] 2020 [cited 2020 Nov 28];11:174-8. Available from: https://www.japtr.org/text.asp?2020/11/4/174/297699




  Introduction Top


The world cosmetics market is estimated that in 2020 the cosmetics market grew to 335.6 billion US dollars[1] Nowadays, the data showed that the product has the ability to have antioxidant and anti-aging properties are growing trend.[2] The medicinal plants are enriched sources of antioxidants properties.[3],[4]Centella asiatica L. is shown high polyphenol and triterpenes such as asiaticoside, madecassoside, and asiatic acid, it was also claimed to be useful in wound healing properties.[5],[6],[7] Many parts of Nelumbo nucifera has various biological components[8] It exhibits several pharmacological effects contain antioxidant and astringent properties.[9]Hibiscus sabdariffa L., the calyces extract, is the high content of anthocyanins,[10] which presents compounds with anti-inflammatory properties.[11] All of the above has shown that Thai edible plants are a potential source of therapeutic bioactive. This research aims to study the phytochemical compositions and bioactive activities of these plants. The results can provide a useful reference for use as anti-aging active ingredients for cosmetic and pharmaceutical industrials.


  Materials and Methods Top


Plant material

C. asiatica aerial part, H. sabdariffa flower, and N. nucifera petal were purchased from Thai Herbal Pharmacy Shop in Pathumthani, Thailand.

Chemicals

2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis (3-ethylbenzothiazoline-6-sunfonic acid) (ABTS), ascorbic acid, Clostridium histolyticum collagenase type I, N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA), porcine pancreas elastase type I, N-Succinyl-Ala-Ala-Ala-p-nitroanilide (AAAPVN), epigallocatechin gallate (EGCG), trizma® base, and potassium persulfate were purchased from Sigma–Aldrich; Merck for dimethyl sulfoxide.

Extraction

All samples were washed with water, ground into powder, and then dehydrated by hot air oven at 50°C. The solvent extracts were prepared by adding 100 g of each sample powder to 1 L of 95% ethanol (1:10 w/v), applying ultrasound-assisted extraction as frequency 40 kHz at 40°C for 30 min then maceration until 7 days, therefore, using a rotary evaporator to remove the solvent and collected crude extract in −20°C consequently the percentage yield calculate.[12]



Gas chromatography–mass spectrometry analysis

The phytochemical constituent present in the ethanolic extracts of C. asiatica, N. nucifera and H. sabdariffa were determined by gas chromatography–mass spectrometry equipped with HP5MS capillary column (30 m × 250 μm × 0.25 μm). The 2 μL of sample volume for injection. In the assay conditions, carrier gas was used ultra-high purity helium and flow rate 1.2 mL/min, split ratio 20:1, thus the inlet temperature at 250°C; consequently, MS programs scanned a quality range of 40–550 amu, solvent delay 3 min, transfer line 280°C.[13] Finally, the identification of the compounds by comparing them with the database of the US NIST mass spectra library.

2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay

The DPPH assay of the extract was modified method as previously described.[14] The 0.2 mM DPPH solution was prepared in absolute ethanol. The solution was added with 100 μL of the tested samples and DPPH solutions. The reaction was determined at wavelength 520 nm after incubated in the darkroom for 30 min; the percent inhibition of radical scavenging activity was calculated as follows Eq. (1):



Moreover, the result was expressed as IC50 value.

2,2'-azinobis (3-ethylbenzothiazoline-6-sunfonic acid) free radical scavenging assay

The ABTS scavenging assay was measured following the modified method by Kim et al.[15] The ABTS reagent was mixed with 7 mM ABTS and 2.45 mM potassium persulfate (8:12 v/v ratio) and incubated in the darkroom temperature for 16 h. The working solution was diluted to 0.70 ± 0.085 OD at 750 nm with water. In addition of 180 μL of the working ABTS solution was mixed with 20 μL of the test sample, the mixture was incubated at room temperature for 30 min, and then absorbance was measurement at 750 nm. The percentageof ABTS radical scavenging was calculated as: (1). Moreover, the result was expressed as an IC50 value.

Anti-collagenase activity

The collagenase activity was modified method by Liyanaarachchi et al.[16] The reaction mixture contained 20 μL samples and 20 μL enzyme solution (0.8 units/mL dissolved in 50 mM Tricine buffer solution, pH 7.5 which was incubated at 37°C for 10 min before use) was incubated for 10 min after adding 200 μL FALGPA substrate (0.5 mM dissolved in buffer) the absorbance was immediately determined at 340 nm at the time interval of 0 and 1 min. Finally, enzymatic activity was assessed by decreased kinetic absorbance which the percentage of collagenase inhibition was calculated as follows:



Anti-elastase activity

The elastase inhibition assay was a method modified by Panjapa and Bungorn.[17] Elastase 2 Units/mL dissolved in 1 M Tris-HCL buffer (pH 8) then incubated at 37°C for 10 min and the substrate AAAPVN was also dissolved in buffer at 1.6 mM. Briefly, the mixture of 40 μL test extracts (mg/mL) and 20 μL enzyme solutions was incubated for 10 min; therefore, add substrate solution 540 μL. Using EGCG as standard, the absorbance was immediately determined at 410 nm at time interval of 0 and 5 min. In the end, the elastase inhibitory effect of test extracts was expressed by the following Eq. (2).

Statistical analysis

The data were showed in triplicate as means ± standard deviation. The statistically significant was analyzed using one-way ANOVA at P < 0.05. Moreover, statistical analysis was performed with GraphPad Prism version 5.01 (San Diego, CA, USA).


  Results and Discussion Top


Extract yield

The percentage ethanolic extract yield of C. asiatica, N. nucifera, and H. sabdariffa were presented 11.53, 13.28, and 27.50, respectively. The differences in percent yield from the sample plants in the present analysis may be determined the availability of different extracted components, which is a result of the chemical composition of various plants.[18]

Gas chromatography–mass spectrometry analysis

The chromatographic ion spectrum of C. asiatica contained 10 peaks indicating the presence of 7 chemical constituents. Nevertheless, Ethanol, 2-(9-octadecenyloxy)-, (Z)-giving the highest peak. N. nucifera were classified 30 peaks signify of 25 components by means of the results exhibited that the capacity of γ-Sitosterol in N. nucifera was the maximum while the substance Ethanol 2-(9 Octadecenyloxy)-(Z)-is found similarly in C. asiatica, the spectrum was described 18 peaks and 15 bioactive constituents, in the present investigation, a variety of compounds have been detected in H. sabdariffa that Hexadecanoic acid shows the maximum peak. Three different extracts show that Ethanol, 2-(-Octadecenyloxy), γ-Sitosterol and Hexadecanoic acid, ethyl ether are representative of the main interest. Especially, Ethanol, 2-(-Octadecenyloxy) is biochemical compounds because it has a good application for pharmacological actions as anti-cancer.[19] The γ-Sitosterol has been reported from soya which have the benefit of anti-diabetic activity.[20] And finally, the extracts of H. sabdariffa showed the presence of Hexadecanoic acid, ethyl ester which has the antioxidant and hemolytic 5-alpha reductase inhibitor feature.[21] Consequently, these compositions may be use as ingredients for the cosmetic and pharmaceutical industrials.

2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity

The scavenging activity of C. asiatica, N. nucifera and H. sabdariffa extracts against DPPH [Figure 1]. The results exhibited that IC50 values are 0.32 ± 0.01, 0.34 ± 0.00 and 0.35 ± 0.01 mg/mL, respectively, and standard ascorbic acid in this assay was 0.005 ± 0.00 when comparing the antioxidant activity of all plants, the result shown that the extract of C. asiatica is high significantly effect (P < 0.05). Corresponding to this result, reported the ethanolic extract of C. asiatica contains the highest phenolic content, which presented the great value of antioxidant more than hexane and chloroform extracts.[5]
Figure 1: Antioxidant activity determined by 2,2-diphenyl-1- picrylhydrazyl method, the tested samples show a significant difference in Centella asiatica and Hibiscus sabdariffa similar to Centella asiatica and Nelumbo nucifera at P < 0.05 (∗)

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2,2'-azinobis (3-ethylbenzothiazoline-6-sunfonic acid) scavenging activity

The ABST scavenging activity of C. asiatica, N. nucifera and H. sabdariffa extracts inhibit ABTS [Figure 2]. The results showed that IC50 values are 0.71 ± 0.02, 0.93 ± 0.06 and 0.62 ± 0.12 mg/mL, respectively, and ascorbic acid were 0.033 ± 0.001, after assessing the antioxidant activity of all plants, the result shown that the extract of H. sabdariffa significantly (P < 0.05) is the finest effect. This result agrees with this reported that ethanolic extract of H. sabdariffa had great antioxidant activity.[22],[23] Although the effect of antioxidant tests on the DPPH and ABTS methods of the three plants will provide different results. In order that it may be due to the rapidity electron transfer during reactions and the phytochemical composition of plants with different antioxidant effects or from other factors, for example, light protection, reaction quality, cleaning, etc.
Figure 2: Antioxidant activity determined by 2,2'-azinobis (3-ethylbenzothiazoline-6-sunfonic acid) method, the tested samples show a significant difference in Centella asiatica and Nelumbo nucifera at P < 0.01 (∗∗) during Nelumbo nucifera and Hibiscus sabdariffa at P < 0.05 (∗)

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Anti-collagenase activity

The ethanolic extract of H. sabdariffa has an excellent collagenase inhibition with a percentage of 90.63 ± 0.00. N. nucifera and C. asiatica also showed anti-collagenase activity with the percentage of 85.94 ± 2.21 and 78.13 ± 4.42, respectively. The results are presented in [Figure 3]. The EGCG was used as standard, and it showed lower activity than all plants at 71.88 ± 0.00. Nevertheless, the effect of collagenase inhibition of ethanolic extracts might involve to different mechanisms. It has been reported that phytochemical compositions are mainly responsible for collagenase inhibition. Besides, collagen is an ample structural protein and extracellular matrix components, which are degraded by collagenase that is a zinc-containing multi-domain enzyme.[24] In conclusion, collagen fibers will decrease with age and damage from UV rays that cause wrinkles and sagging. Consequently, all of these extracts have possible uses as anti-aging ingredients in health products like dietary supplements or cosmetics.
Figure 3: Anti-collagenase of three extracts, the tested-samples show a significant difference in Centella asiatica and Hibiscus sabdariffa at P < 0.01 (∗∗) while Nelumbo nucifera and Hibiscus sabdariffa at P < 0.05 (∗)

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Anti-elastase activity

The anti-elastase activity assay performed by taking EGCG as a standard, showed that C. asiatica with the great elastase inhibition of these plants that percentage of 12.45 ± 0.44. N. nucifera and H. sabdariffa also presented anti-elastase activity with a percentage of 6.67 ± 0.15 and 1.29 ± 0.91, respectively. The results are presented in [Figure 4]. However, their inhibitions were lower than the standard EGCG, with the percentage of 95.89 ± 0.22. Elastin is a 2%–4% insoluble fiber protein found in the dermis matrix, which plays an important role in skin elasticity. In contrast, it is often degraded by elastase enzyme, which can be hydrolyzed on the peripheral proteins and structures in connective tissues.[13] Although, the experimental results show a low elastase inhibition rate (12.45 ± 0.44 mg/mL) when compared to the standard. However, there are still observations that medicinal plants are lower price than synthetic chemicals sold in the market. It also gives a feeling of safety for customers because it is a natural ingredient. Therefore, the results suggested that C. asiatica can be used as a raw material to resist the enzyme elastase but may need to be used at higher concentrations for improved qualifications.
Figure 4: Anti-elastase of three extracts, the test sample shows a significant difference in Centella asiatica and Nelumbo nucifera or Hibiscus sabdariffa at P < 0.001 (∗∗∗) while Nelumbo nucifera and Hibiscus sabdariffa at P < 0.001 (∗∗∗)

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  Conclusion Top


Our results indicate the various bioactive contents of C. asiatica, N. nucifera, and H. sabdariffa ethanolic extracts have an interest function for pharmacological actions such as antioxidant capability due to its good ability to resist DPPH and ABTS free radical scavenging activities (IC50< 0.93) and total extract are great capable of inhibiting collagenase (inhibiting collagenase more than 78.13%), but elastase is poorly inhibited especially from H. sabdariffa. Consequently, these edible plants can use as an anti-aging active ingredient for cosmetic and pharmaceutical industrials.

Financial support and sponsorship

This research was financially supported by Institute of Research and Development, Rajamangala University of Technology Thanyaburi, Thailand (Project code: IRF62A1204). We are thankful to Thai Traditional Medicine College for providing the laboratory facilities.

Conflicts of interest

There are no conflicts of interest.



 
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