Abstract
This current work aims to determine phytochemicals, in vitro radical scavenging, and in vivo oxidative stress reduction activities of peppermint (Mentha piperita L.) ethanolic extract (PEE). The Clule method was used to determine the phytochemical content. An in vitro antioxidant with radical scavenging activity was measured using 2,2-diphenyl-1-picrylhydrazyl. An in vivo antioxidant with oxidative stress reduction was carried out for 10 days on 25 male Sprague–Dawley rats (divided into five groups). Every day, each group was given positive control, negative control, 5, 10, and 20 mg/200 gr of body weight (BW) of the extract. The blood plasma was taken for malondialdehyde analysis. A phytochemical identification of PEE revealed more compounds, such as flavonoids, alkaloids, steroids, essential oils, and tannin. PEE exhibits significant in vitro radical scavenging activity, with an IC50 value of 126.695 µg/mL. In the in vivo antioxidant with oxidative stress reduction experiments, 5 mg/200 gr BW was the most effective dose, as evidenced by a considerable drop in malondialdehyde level (0.312 nmol/mL) after and before treatment. In conclusion, PPE has the potential to be developed as a herbal antioxidant based on in vitro and in vivo test results.
Keywords: Antioxidant, fifty percent inhibition concentration, malondialdehyde level, peppermint (Mentha piperita L.) extract, phytochemical
INTRODUCTION
Degenerative diseases are still the most prevalent cause of death in Indonesia. In 2014, the World Health Organization (WHO) mentioned that the total deaths in Indonesia were 1.551.000 and that about 71% of them were because of degenerative diseases or noncommunicable diseases (NCDs). The most considerable percentage of NCDs was cardiovascular diseases with 37%, higher than other communicable, maternal, perinatal, and nutritional conditions with 22%. The rest of the causes are cancers, other NCDs, diabetes, and chronic respiratory diseases, with a percentage of 13%, 10%, 6%, and 5%, respectively.[1]
The main problems in degenerative diseases are damaged healthy cells in our bodies, caused by free radicals. These damaged cells make them unable to work correctly as their function and structure. These free radicals originate from reactive oxygen species (ROS), reactive oxygen atoms with an unstable electrical charge or those with an unpaired electron, thus finding another electron but making them unstable. When ROS attacks the normal functional cells, causing them to be damaged, this condition is known as oxidative stress. Oxidative stress is a shift in the balance between prooxidants and antioxidants inside our bodies.[2]
Consuming antioxidants to counteract pro-oxidants can be gained by endogenous antioxidants and exogenous antioxidants (dietary antioxidants).[3] One of the most considerable resources for those antioxidants are plants, among which peppermint (Mentha piperita L.) (PEE) can be gained quickly in Indonesia. Therefore, research is needed to prove the antioxidant effect of peppermint leaves in Indonesia.
The antioxidant substances are polyphenols and volatile phenolic oils, without including the in vivo assay on animal models.[4] Thus, it raises a question about whether the antioxidant substances present in peppermint can neutralize free radicals based on the in vitro 2,2-diphenyl-1-picrylhydrazyl (DPPH) test and compensate for the in vivo oxidative stress using Sprague–Dawley rats at its most effective dosage. It is hypothesized that peppermint has an exogenous antioxidant effect on in vitro free radicals (IC50 value) and in vivo oxidative stress (malondialdehyde level).
The present study examines the effect of peppermint leaves as an herbal antioxidant and how effective it is in vivo and in vitro. This research is expected to guide and reference future clinical practice regarding the health problems caused by free radicals.
MATERIALS AND METHODS
Preparation of peppermint (mentha piperita L.) ethanol extract
The leave of peppermint sample was achieved from Balai Penelitian Tanaman Rempah dan Obat (Balittro) with specimen voucher No. 056/BPTRO-RR/FK-UI/VIII/2019, Bogor, West Java, Indonesia, in August 2019. The sample specimen was authenticated and identified at the Indonesian Institute of Sciences – Cibinong, West Java, Indonesia. The extraction method used maceration at room temperature using 70% ethanol. The extract solution was evaporated to obtain a thick extract (50 rpm, 30°C–40°C).[5]
Phytochemical identification
A phytochemical identification was conducted to identify secondary metabolites (mainly the antioxidant compound) in PEE.[6] The aforementioned concentrated extract was separated using three polarity solvents (n-hexane, ethyl acetate, and 70% ethanol). The qualitative phytochemical constituent analysis was used in the standard procedures described by Ambarwati et al.[7]
In vitro activity assay of 2,2-diphenyl-1-picrylhydrazyl radical scavenging
The DPPH radical scavenging test is one of the most widely utilized techniques to assess the antioxidant activity of certain substances. The in vitro test is based on the Yen and Chen method[8] to measure the IC50. Then, to calculate the IC50, linear regression analysis should be done by converting the result of inhibition percentage into a linear equation. As a result, the formula (y = ax + b) is formed with the y-axis as the inhibition percentage and the x-axis as the extract concentration.[9] We used quercetin as a positive control.
In vivo malondialdehyde level-oxidative stress reduction activity assay
This study is an analytical experimental approved (the ethical approval number is 0070/UN2. F1/ETIK/2018) by the ethical committee of the Faculty of Medicine, Universitas Indonesia. This study employed 25 mature male Sprague-Dawley rats (weighing 180–200 g). Some different dosages of extract were used, including 5, 10, and 20 mg/200 g BW/day, Vitamin C as a positive control (2 mg/200 g BW/day). They were given for 10 days after the first blood puncture. A blood sample and a malondialdehyde test (500 µL) were taken with a capillary pipe in the sinus orbitalis canthus and placed in a tube with heparin at two moments, before and after the rat was given both physical exercise and PEE. The blood samples were centrifuged to get the plasma. The clear filtrate at the top, the plasma, was transferred to another tube and kept at-20°C.
Malondialdehyde determination was performed based on the literature.[10] Firstly, 250 µL blood plasma was mixed with 100 µL of 8.1% sodium dodecyl, 750 µL of thiobarbiturates (20 M), and 750 µL of HCl (0,5 M), and 125 µL of aquabidest. Then, it was mixed using a vortex. After that, the mixture was heated at a temperature of 90°C for 15 min. Then, it settled and cooled down for 10 min. The next step was to add 500 µL aquabidest and 2.5 mL n-butanol to the mixture, vortex it, and then centrifugated at 3000 rpm. Finally, the filtrate was placed in a fluorometer, and the absorbance at 520 nm was measured. To compute the malondialdehyde level, 1,1 33-tetra-ethoxy-propane (with some concentration of 5, 10, and 20 nmol/mL) was employed in conjunction with the malondialdehyde standard curve (linear equation) by comparing malondialdehyde concentration in the y-axis with absorbance in the x-axis.[10]
Data analysis and processing plan
The in vitro (IC50 value) and in vivo antioxidant activity experiments were evaluated using linear regression analysis with IBM SPSS statistic 24 software (IBM Corp., USA). A paired t-test and a one-way ANOVA test were both performed. To compare the Malondialdehyde levels after and before the physical and antioxidant activity, the paired t-test was utilized. The one-way ANOVA test was performed to examine the differences and efficiency of the PEE and Vitamin C as positive controls. The Shapiro-Wilk test was used to determine a normal data distribution. The post hoc Bonferroni test was used for homogeneity of variances.
RESULTS
Phytochemical identification
The phytochemical identification result of fractions from PEE was demonstrated in Table 1.
Table 1.
The phytochemical identification of some fraction from peppermint (Mentha piperita L.) ethanolic extract
| Secondary metabolite | n-Hexane fraction | Ethyl acetate fraction | Ethanol fraction |
|---|---|---|---|
| Tannin | − | + | + |
| Saponin | − | + | − |
| Terpenoid/steroid | − | − | + |
| Alkaloid | − | + | + |
| Glycosides | − | − | − |
| Flavonoid | − | + | + |
| Essential oil | − | + | + |
+: Positive, -: Negative
In vitro 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity of PEE
As shown in Table 2, the higher the concentration of quercetin and the sample, the better the activity exerted. The linear regression equation for quercetin was y = 1.4331x + 29.686; R2 = 0.99165. While the R2 value was used to determine an adequate correlation between quercetin concentration and DPPH inhibition. The IC50 value was calculated by inserting 50 as the value of “y,” resulting in 14.17 µg/mL of quercetin concentration needed to inhibit 50% of the DPPH radical. Moreover, antioxidant activity of PEE was calculated with the equation as follows y = 0.3983× +0.4567; R2 = 0.99834. After calculating, the IC50 of PEE was 126.695 µg/mL.
Table 2.
IC50 value of peppermint (Mentha piperita L.) ethanolic extract, based on 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay
| Sample | Concentratio (µg/mL) | Inhibition (%) | IC50 (µg/mL) |
|---|---|---|---|
| Quercetin | 1 | 4.72 | 14.17 |
| 5 | 39.23 | ||
| 10 | 73.68 | ||
| 50 | 96.70 | ||
| PEE | 10 | 2.85 | 126.695 |
| 50 | 18.99 | ||
| 100 | 41.36 | ||
| 200 | 78.36 |
DPPH: 2,2-diphenyl-1-picrylhydrazyl, PPE: Peppermint (Mentha piperita L.) ethanolic extract, IC50: fifty percentage inhibition concentration
In vivo malondialdehyde level-oxidative stress reduction activity of PEE
The difference in malondialdehyde level was indicated in Table 3, where all groups, including the positive control, saw a substantial drop in malondialdehyde level following treatment.
Table 3.
Malondialdehyde level
| Groups | Before treatment (nmol/mL) | After treatment (nmol/mL) | Differences (nmol/mL) |
|---|---|---|---|
| PEE dose of 5 mg | 1.228 | 0.735 | −0.493 |
| PEE dose 10 mg | 1.272 | 0.874 | −0.398 |
| PEE dose 20 mg | 1.012 | 0.772 | −0.241 |
| Negative control | 0.823 | 0.974 | +0.151 |
| Positive control | 0.889 | 0.678 | −0.210 |
PPE: Peppermint (Mentha piperita L.) ethanolic extract
Table 4 shows the outcome of the paired t-test, which indicated the relationship between malondialdehyde levels before and after therapy. The P < 0.05 suggests that there is a statistically significant difference between at least two data groups.
Table 4.
The paired t-test result of malondialdehyde level
| Malondialdehyde level of groups | Total subjects | Mean (nmol/mL)±SD | Mean difference (nmol/mL)±SD | 95% CI of the difference | P |
|---|---|---|---|---|---|
| Before treatment | 25 | 1.075±0.182 | 0.299±0.177 | 0.226–0.372 | 0.000 |
| After treatment | 25 | 0.776±0.101 |
SD: Standard deviation, CI: Confidence interval
The one-way ANOVA test [Table 5] was employed to determine a statistically significant amount of variation of two or more unconnected elements. It also determines the most effective PEE dose for reducing oxidative stress.
Table 5.
The one-way ANOVA result of malondialdehyde level
| Group (total subject) | Mean (nmol/mL)±SD | P |
|---|---|---|
| PEE dose of 5 mg (5) | 0.493±0.072 | 0.003 |
| PEE dose of 10 mg (5) | 0.398±0.119 | |
| PEE dose of 20 mg (5) | 0.241±0.134 | |
| Positive control (5) | 0.210±0.416 | |
| Negative control (5) | 0.151±0.219 |
SD: Standard deviation, PPE: Peppermint (Mentha piperita L.) ethanolic extract
The following test was the post hoc test with Bonferroni Correction [in Table 6], where each component was compared to other components in the same group. There were several significant comparison values (P < 0.05) between components in the group, including rats given 5 mg of extract and a positive control group with 0.029, meaning that it was more significant when applying 5 mg rather than the positive control. PEE (5 mg) with a negative control group with 0.006, meaning that it was more significant when applying 5 mg rather than the negative control.
Table 6.
The post hoc test result
| Groups 1 | Groups 2 | Significance |
|---|---|---|
| PEE dose of 20 mg | PEE dose of 10 mg | 0.739 |
| PEE dose of 5 mg | 0.067 | |
| Positive control | 1.000 | |
| Negative control | 1.000 | |
| PEE dose of 10 mg | PEE dose of 20 mg | 0.739 |
| PEE dose of 5 mg | 1.000 | |
| Positive control | 0.358 | |
| Negative control | 0.077 | |
| PEE dose of 5 mg | PEE dose of 20 mg | 0.067 |
| PEE dose of 10 mg | 1.000 | |
| Positive control | 0.029 | |
| Negative control | 0.006 | |
| Positive control | PEE dose of 20 mg | 1.000 |
| PEE dose of 10 mg | 0.358 | |
| PEE dose of 5 mg | 0.029 | |
| Negative control | 1.000 | |
| Negative control | PEE dose of 20 mg | 1.000 |
| PEE dose of 10 mg | 0.077 | |
| PEE dose of 5 mg | 0.006 | |
| Positive control | 1.000 |
PPE: Peppermint (Mentha piperita L.) ethanolic extract
DISCUSSION
Phytochemical identification was made by fractionating the extract with ethanol, ethyl acetate, and n-hexane through several chemical reactions to determine the content of PEE as an antioxidant. Most of the antioxidant compounds, saponin, alkaloid, flavonoid, essential oil, tannin, and triterpenoid/steroid, are present in the ethanol fraction due to the polar solvent. However, because hexane was a nonpolar solvent, it did not contain any phytochemical compounds. In contrast, ethyl acetate was a semi-polar solvent that contained saponin, alkaloids, flavonoids, essential oils, and tannin.
Previous studies about the phytochemical profile of peppermint have been conducted by Ji et al., who stated that the major constituents of the antioxidant substance were essential oil and terpenoid.[11] Based on a study conducted by Lisawati and Sulianti (2002), the main antioxidant constituents are flavonoids and essential oils.[12] The other study by Thantsin et al. showed that flavonoids, terpenoids, and essential oils are the major antioxidant substances in peppermint.[13] According to the aforementioned references, the most probable antioxidant substances that exert antioxidant activity are flavonoids, essential oils, and terpenoids.
In order to obtain the data, the antioxidant activity of PEE was compared with quercetin (positive control). Then, DPPH radical scavenging was given to evaluate the IC50 value, the extract concentration needed to decrease the oxidative stress level by 50%. A small value of the IC50 level indicates intense antioxidant activity.[14,15,16] Based on the comparison of antioxidant activity, antioxidant intensity was categorised such as strong (IC50 <50 µg/mL), active (IC50 50–100 µg/mL), moderate (IC50 101–250 µg/mL), weak (IC50 251–500 µg/mL), and inactive (IC50 >500 µg/mL).[17,18] From the result mentioned above, the IC50 value of PEE is categorized as moderate antioxidant intensity.
Malondialdehyde was defined as the lipid peroxidation product in the long chain of unsaturated fatty acids in the lipid membrane. Lipid peroxidation happens when the oxidative stress level increases. Thus, this indicator was used to determine the antioxidant activity, which, in this case, was PEE, to decrease the oxidative stress level.[19] The paired t-test was used to assess the comparison. The P = 0.005, which was <0.05, showing a significant change in malondialdehyde levels before and after treatment.[20]
CONCLUSION
The phytochemical compounds of PEE were saponin, flavonoids, alkaloids, terpenoids, essential oils, and tannin. The effect of PEE in compensating for oxidative stress was evident in vitro, as shown by the IC50 value of 126.695 µg/mL, which is categorized as moderate antioxidant intensity. By comparing the extract concentration with the malondialdehyde level differences before and after treatment, the most effective dosage was 5 mg/200 gr BW, which decreased the malondialdehyde level by 0.492 nmol/mL.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgment
We would like to express our gratitude to International Indexed Publication Grant for Students Final Projects from the University of Indonesia 2018 which have supported this research.
REFERENCES
- 1.World Health Organization (WHO) Non-Communicable Diseases and Mental Health Country Profiles Indonesia; 2014. [Last accessed on 2021 Dec 16]. Available from: http://www.who.int/nmh/countries/idn_en.pdf .
- 2.Percival M. Washinton DC: Clinical Nutrition Insights; 1998. [Last accessed on 2021 Dec 16]. Antioxidants. Available from: https://acudoc.com/Antioxidants. PDF . [Google Scholar]
- 3.Bhattacharya S. Reactive oxygen species and cellular defense system. In: Rani V, Yadav UC, editors. Free Radicals in Human Health and Disease. New Delhi: Springer; 2015. pp. 17–29. [Google Scholar]
- 4.Ervina M, Nawu YE, Esar SY. Comparison of in vitro antioxidant activity of infusion, extract, and fractions of Indonesian cinnamon (Cinnamomum burmannii) bark. Int Food Res J. 2016;23:1346–50. [Google Scholar]
- 5.Ambarwati NS, Ahmad I, Elya B, Malik A, Hanafi M. Pharmacognostic and antimicrobial studies of Garcinia latissima Miq.leaves (Clusiaceae) Pharmacogn J. 2017;9:493–8. [Google Scholar]
- 6.Clule I. Methodology for analisys of vegetable drugs, practical manuals on industrial utilization of medicinal and aromatic plant. Phytochemistry. 1982;63:97–104. [Google Scholar]
- 7.Ambarwati NS, Elya B, Malik A, Hanafi M. Phytochemical and antimicrobial studies on Garcinia lattissima Miq.fruit extract. Asian J Pharm Clin Res. 2017;10:230–2. [Google Scholar]
- 8.Yen GC, Chen HY. Antioxidant activity of various tea extracts in relation to their anti-mutagenicity. J Agric Food Chem. 1995;43:27–32. [Google Scholar]
- 9.Ambarwati NS, Elya B, Sa’adah AN, Puspitasari N, Malik A, Hanafi M. Activity of fractions from Garcinia latissima Miq.leaves ethyl acetate extracts as antibacterial against Bacillus subtilis and antioxidant. Adv Sci Lett. 2018;24:6366–70. [Google Scholar]
- 10.Abdelghffar EA, El-Nashar HA, Al-Mohammadi AG, Eldahshan OA. Orange fruit (Citrus sinensis) peel extract attenuates chemotherapy-induced toxicity in male rats. Food Funct. 2021;12:9443–55. doi: 10.1039/d1fo01905h. [DOI] [PubMed] [Google Scholar]
- 11.Ji X, Pu QL, Garraffo HM, Pannell LK. Essential oils of the leaf, bark and branch of Cinnamomum burmannii Blume. J Essent Oil Res. 1991;3:373–5. [Google Scholar]
- 12.Lisawati Y, Sulianti SB. The influence of distillation time and powder size on cinnamylaldehyde content of the volatile oil of bark of Cinnamomum burmanii Nees. Indones J Pharm. 2002;13:123–32. [Google Scholar]
- 13.Thantsin K, Zhang Q, Yang J, Wang Q. Composition of semivolatile compounds of 10 Cinnamomum species from China and Myanmar. Nat Prod Res. 2008;22:576–83. doi: 10.1080/14786410701592802. [DOI] [PubMed] [Google Scholar]
- 14.Roy A, Bhoumik D, Sahu RK, Dwivedi J. Phytochemical screening and antioxidant activity of Sesbania grandiflora leaves extracts. Asian J Res Pharm Sci. 2014;4:16–21. [Google Scholar]
- 15.Raaman N, Selvarajan S, Balakrishnan D, Balamurugan G. Preliminary phytochemical screening, antimicrobial activity and nutritional analysis of methanol extract of Asparagus racemosus (Willd) roots. Res J Pharm Tech. 2009;2:777–9. [Google Scholar]
- 16.Tiwari P. Phenolics and flavonoids and antioxidant potential of balarishta prepared by traditional and modern methods. Asian J Pharm Anal. 2014;4:5–10. [Google Scholar]
- 17.Rosmalena R, Widyastuti PA, Yazid F, Ambarwati NS, Ahmad I. Phytochemicals and antioxidant activity evaluation of Origanum vulgare (L.) stem bark extracts. Pharmacogn J. 2021;13:965–70. [Google Scholar]
- 18.Dash S, Sahoo AC, Mishra B. In vitro and in vivo antioxidant assessment and hepatoprotective activity of Aponogeton natans (Linn.) Engl. and Krause on diclofenac sodium induced liver toxicity. Res J Pharm Tech. 2018;11:4431–8. [Google Scholar]
- 19.Catalá A. A synopsis of the process of lipid peroxidation since the discovery of the essential fatty acids. Biochem Biophys Res Commun. 2010;399:318–23. doi: 10.1016/j.bbrc.2010.07.087. [DOI] [PubMed] [Google Scholar]
- 20.Islam MR, Islam MR, Ahmed I, Al Moktadir A, Nahar Z, Islam MS, et al. Elevated serum levels of malondialdehyde and cortisol are associated with major depressive disorder: A case-control study. SAGE Open Med. 2008;6:1–7. doi: 10.1177/2050312118773953. [DOI] [PMC free article] [PubMed] [Google Scholar]