Hibiscus sabdariffa tea improved the maximum oxygen uptake (VO 2 max) and blood pressure in normotensive participants

The heavy physical practice may escalate the production of reactive oxygen species, which eventually results in cell damage. Previous studies announced that treatment with antioxidants might decrease oxidative stress caused by physical practice. Studies on the ethanol and water extracts of Hibiscus sabdariffa calyces had proven their antioxidant activity. Our work is the first report on the effect of 30 days of daily intake of H. sabdariffa tea on the maximum oxygen uptake (VO 2 max) and blood pressure (BP) in healthy male and female normotensive participants. The VO 2 max, measured to obtain the participants’ cardiovascular fitness status, was performed by following the Young Men’s Christian Association step test. Our study revealed that H. sabdariffa tea intake decreased the BP of the participants in week 4 and increased the number of participants with average VO 2 max status, starting from week 2 to 4, compared to baseline. Taking these results together, H. sabdariffa tea may be beneficial to improve the maximum oxygen uptake status in normotensive participants.


INTRODUCTION
Hibiscus sabdariffa L. has been widely explored in both healthy participants and patients with metabolic disorders. Hibiscus sabdariffa has indicated a good effect on hypertensive patients (Herrera-Arellano et al., 2007;McKay et al., 2010), as proven by a randomized double-blind clinical study of H. sabdariffa L. (containing 250 mg of total anthocyanins/dose) administered for 4 weeks. This study resulted in a decrease in blood pressure (BP) (Herrera-Arellano et al., 2007). Another randomized double-blind clinical study on 65 participants with pre-and mild hypertension treated with brewed H. sabdariffa L. tea reported a significantly lower systolic BP, but not significantly lower diastolic BP (McKay et al., 2010). A study on type II Diabetes mellitus patients with mild hypertension consuming H. sabdariffa L. infusion demonstrated positive effects on their BP (Mozaffari-Khosravi et al., 2009). In a randomized control trial, patients with nonalcoholic fatty liver disease treated with a 450 mg capsule of H. sabdariffa or a placebo capsule daily for 8 weeks experienced a decrease in serum triglyceride, alanine aminotransferase, and aspartate aminotransferase levels compared with the placebo. A significant decrease in systolic BP and diastolic BP and a significant elevation in total antioxidant capacity had also been described (Izadi et al., 2021). Moreover, a rise in high-density lipoprotein cholesterol and a significant decrement in BP of healthy participants treated with H. sabdariffa tea for 30 days were also reported (Diantini et al., 2021).
The physical practice is believed to be the foundation of good health; however, this activity may escalate the production of ROS, which eventually results in cell damage. Moreover, physical practice results in increasing the levels of malondialdehyde in the blood which works as an indirect indicator of lipid peroxidation (Clarkson and Thompson, 2000). The physical practice is believed to be able to stimulate inflammation and muscle fatigue (Simioni et al., 2018). Also, during heavy physical practice, the oxygen flux to active skeletal muscles is significantly elevated (Atalay et al., 2015;Radak et al., 2013). The demand for O 2 in skeletal muscle during physical workups leads to a remarkable change in blood flow (Kawamura and Muraoka, 2018). Previous studies announced that treatment with antioxidants might decrease oxidative stress caused by heavy physical practice (Clarkson and Thompson, 2000). Participants supplemented with vitamin E had shown a decrease in creatine kinase and lactic dehydrogenase levels which confirmed the protective effect of vitamin E against oxidative stress (Itoh et al., 2000).
Considering this, it is interesting to explore the effect of H. sabdariffa, which has been reported for its ability to increase the activity of antioxidant enzymes, on normotensive participants' post-physical practice in terms of cardiovascular fitness. Thus, our work assessed the effect of 30 days of daily intake of H. sabdariffa tea on the VO 2 max and BP in healthy participants. VO 2 max equals the maximal O 2 consumption during exercise to approximately the depletion of physical strength. It is one of the best methods of predicting cardiorespiratory endurance and is related to body weight (Blair et al., 2001).

Study design
This study was a quasi-experiment with a pre-post design for 32 adult males (59.4%) and females (40.6%), aged 21-55 years. The participants were affirmed to be healthy by a physician. Participants diagnosed with hypertension, peptic ulcers, knee arthritis, and/or musculoskeletal disorders were excluded. The protocol of this study was approved by the Research Ethics Committee of Universitas Padjadjaran (approval no. 35/UN6.C.10/ PN/2018). The participants had agreed to sign the informed consent in compliance with the Ethical Principles for Medical Research Involving Human Subjects of the WMA Declaration of Helsinki (https://www.wma.net/policies-post/wma-declarationof-helsinki-ethical-principles-for-medical-research-involvinghuman-subjects/) with the knowledge that they could withdraw at any time.
The general characteristics of the participants based on their age and body mass index (BMI) are shown in Tables 1 and 2, respectively. The participants were given 200 ml of H. sabdariffa tea, twice daily for 30 successive days. The H. sabdariffa tea was prepared by boiling 100 g of dried calyces and 500 g of sugar in 10 l of potable water for 15 minutes; thus, the daily dose of the H. sabdariffa tea (2 × 200 ml) contains approximately 4 g of dried calyces and 20 g of sugar.

Measurement of VO 2 max and BP before and after treatment with H. sabdariffa tea
Before the intervention and every Friday during the treatment, the participants were required to undertake cardiovascular fitness by precisely obeying the Young Men's Christian Association (YMCA) step test (Beutner et al., 2015;Diantini et al., 2021;Morrow et al., 2005;Teren et al., 2016). The participants were prepared to avoid taking meals, cigarettes, and caffeine for at least 2 hours before the test. The test was conducted as follows.
The BP of participants was measured before the step test. The participants were told to sit for 5 minutes on an upright chair in a position with their feet flat on the floor. BP was measured using a sphygmomanometer and stethoscope (Muntner et al., 2019) with three replicates per reading.
Afterward, the participants were directed to stand facing a bench (about 30 cm in height/Harvard bench). A sensor plate connected to a computer was equipped on their chest to record the heart rate. The operator set the metronome to 96 beats per minute (24 steps per minute). The participants started stepping on and off (right foot up, left foot up, right foot down, and left foot down) the bench with a regular rhythm for 3 minutes. After 3 minutes, the participants stopped and sat on the bench for 1 minute. The VO 2 max was determined by adopting the table described by the American Heart Association Cardiorespiratory Fitness Chart published in 1972. The classification is presented in Table 3 (Herdy and Caixeta, 2016; Morrow et al., 2005).

Statistical analysis
Data were analyzed using the Wilcoxon test in IBM Statistical Package for the Social Sciences Statistics version 20.0 for Windows with a p-value <0.05 indicating a statistical significance. Figure 1 presents the cardiovascular fitness of the participants based on BP and VO 2 max. A decrease in systolic and diastolic BP (p = 0.036 and p = 0.03, respectively) compared to baseline was observed in week 4 (Fig. 1a). A decline in the number of participants with very poor VO 2 max (from 23 participants before intervention to 6 participants at week 4 of intervention) was also observed. However, six participants with very poor VO 2 max status have not changed. Inversely, an increase in the number of participants with average VO 2 max (from 4 participants before intervention to 14 participants at week 3 of intervention) was confirmed. Detailed information on the VO 2 max of all participants from the baseline (before intervention with H. sabdariffa tea) to week 4 is presented in Table 4. Statistical analysis revealed a significant difference in the number of participants with very poor and average VO 2 max after intervention with H. sabdariffa tea (p < 0.05) compared to baseline (Fig. 1b). Therefore, the intervention with H. sabdariffa tea might influence cardiovascular fitness, in terms of the BP and VO 2 max parameters.

DISCUSSION
In our study, H. sabdariffa tea was administered twice daily to 32 healthy normotensive Indonesian participants and its effect on cardiovascular fitness was observed in terms of BP and VO 2 max every week until 4 weeks. Both the systolic and diastolic BP of the participants reduced significantly. Our study is in accordance with previous studies reported elsewhere (Al-Shafei and El-Gendy, 2013;Diantini et al., 2021;Herrera-Arellano et al., 2007;Izadi et al., 2021;McKay et al., 2010;Mozaffari-Khosravi et al., 2009). Moreover, a non-randomized quasi-experimental study in Egypt reported a significant decrement in pulse pressure and BP of the normotensive and hypertensive participants compared to their baseline values by the end of week 4 (Al-Shafei and El-Gendy, 2013).
However, apparently, no human studies on the H. sabdariffa effect on cardiovascular fitness were reported. Our study is the first report on the effect of H. sabdariffa tea on the VO 2 max of normotensive participants. The intake of H. sabdariffa tea twice daily for 30 days could significantly reduce the number of participants with very poor cardiovascular fitness and increase the number of participants with average cardiovascular fitness. Based on these data, H. sabdariffa tea could improve the VO 2 max and BP in normotensive participants.
The calyces of H. sabdariffa have been proven to contain abundant anthocyanins, which is indicated by the dark red color. We predict that these anthocyanins may play important role in improving the cardiovascular fitness of the participants. In fact, several epidemiological studies have described the correlation between anthocyanins and cardiovascular diseases. An earlier cohort study reported that the intake of 0.2 mg/day of anthocyanins (strawberries and blueberries) was associated with a decreased risk of cardiovascular diseases in elderly females (Mink et al., 2007). Anthocyanins intake was also reported to have the ability to prevent chronic inflammatory diseases in men and women (aged 40-74 years) (Karlsen et al., 2007). Generally, VO 2 max is being used as the marker of cardiovascular health status. It is considered an important physiological measure in healthy adults (Bennett et al., 2016). A previous study on Korean women (aged 30-59 years), with more than 30% body fat, reported that systolic and diastolic BP was significantly correlated with VO 2 max (Shin and Ha, 2016). Moreover, in a study on 24 Sweden athletes and 5 healthy nonathletes, no significant correlation between VO 2 max and the systolic BP/VO 2 slope was found, but a significant correlation between VO 2 max and systolic BP in males was observed (Eklund, 2021).

CONCLUSION
Our study assessed the effect of 30 days of H. sabdariffa tea consumption on the VO 2 max and BP in healthy male and female normotensive participants. We declared that this is the first study on normotensive humans assessing the effect of H. sabdariffa tea on their cardiovascular fitness. Hibiscus sabdariffa tea intake could significantly reduce the systolic and diastolic BP of the participants. Moreover, H. sabdariffa tea significantly reduced the number of participants with very poor VO 2 max status and significantly increased the number of participants with average VO 2 max status. Although this study was applied to a limited number of participants (n = 32), we concluded that H. sabdariffa tea could improve the VO 2 max and BP in normotensive participants.

AUTHORS' CONTRIBUTIONS
Ajeng Diantini contributed to conceptualization, methodology, funding acquisition, and supervision. Leonardo Lubis contributed to methodology and data curation. Sri Adi Sumiwi contributed to supervision. Syamsul Rahmat contributed to participant recruitment and clinical investigation. Annisa Alpiani contributed to participant recruitment and clinical investigation. Jutti Levita contributed to writing the original draft, reviewing, editing, and supervision. All the authors have read and approved the final article.

FINANCIAL SUPPORT
This research was funded by Academic Leadership Grant (no. 1373i/UN6.O/LT/2019). The publication fee is funded by Universitas Padjadjaran via the Directorate of Research and Community Engagement.

CONFLICTS OF INTEREST
All the authors declared no potential conflicts of interest.

ETHICAL APPROVALS
The protocol of this study has been approved by The Ethical Committee of Padjadjaran University (document no. 35/ UN6.C.10/PN/2018). Data availability can be accessed from the first author upon request.

DATA AVAILABILITY
All data generated and analyzed are included in this research article.

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