Materials and methods:

Research activities were conducted at the Nutritional Analysis Laboratory of Riphah

International University, Faisalabad, for the development and nutritional assessment of a
functional herbal tea. The study aimed to explore the health benefits of incorporating dried
hibiscus, basil leaves, lemon peel, ginger, green tea, and cinnamon in tea formulation to attenuate
the symptoms of hypertension overall health.

Five different treatments were developed using varying proportions of selected ingredients. The
samples were subjected to proximate analysis, antioxidant activity, determination of total
phenolic content TPC, redical scavenging activity by using DPPH and sensory evaluation was
also conducted to assess consumer acceptability.

Area of Research

This study was performed in the Nutritional Analysis and Composition Lab at Riphah
International University, Faisalabad. The purpose of this research was to develop a functional
herbal tea using natural plant ingredients to minimise the symptoms of hypertention.

Procurement of Raw Materials

Raw materials including hibiscus flowers, basil leaves, lemon, ginger, green tea leaves, and
cinnamon sticks were procured from the local market in Faisalabad. All ingredients were
selected based on freshness, quality, and nutritional value.

Preparation of Dried Hibiscus

Fresh hibiscus flowers were cleaned to remove dust and impurities. The flowers were then dried
using an air dryer at controlled temperature until complete removal of moisture was achieved.
The dried hibiscus was stored in airtight containers for further use.

Preparation of Dried Basil Leaves

Fresh basil leaves were washed thoroughly and air-dried to remove surface moisture. The leaves
were then dried in an air dryer at low temperature to preserve their natural color and bioactive
compounds. The dried leaves were stored in airtight containers.

1
Fresh lemons were washed properly, and the peels were separated manually. The peels were
dried using an air dryer at controlled temperature until crisp and moisture-free. The dried lemon
peels were stored in airtight containers.

Product development:

Treatment Hibiscus % Basil leaves % Lemon peel%

T0 100 0 0
T1 70 20 10
T2 60 25 15
T3 50 30 20

Preparation of Herbal Tea:

The ingredients used for the preparation of herbal tea included dried Hibiscus petals, dried Basil
leaves, and dried Lemon peel. The ingredients were first cleaned to remove dust and any foreign
matter. The dried ingredient was measured according to the treatment plan. The dried hibiscus,
basil leaves, and lemon peel were placed together in a clean teapot or bowl. Boiling water (100
mL) was then poured over the dried ingredients, and the mixture was allowed to steep for 5–7
minutes to extract flavor, aroma, and bioactive compounds. After steeping, the tea infusion was
filtered through a fine sieve to remove the solid residues. The clear herbal tea was then poured
into cups and served warm for sensory evaluation and further analysis, following the procedure
described by (Khan et al., 2014)

Proximate Analysis of Herbal Tea

Each sample of herbal tea was analyzed using standard methods described by the Association of
Official Analytical Chemists for the determination of moisture, crude fiber, crude protein, crude
fat, ash, and nitrogen-free extract (NFE) (AOAC, 2016).

Moisture Content

The moisture content of herbal tea samples was determined using the hot air oven. A known
weight of the dried tea sample was taken in a pre-weighed crucible and placed in a hot air oven
at 105°C for 24 hours. After drying, the sample was cooled in a desiccator for 5–10 minutes and

2
weighed. The process was repeated until a constant weight was obtained, and moisture
percentage was calculated using standard formula (AOAC, 2016).

The percentage moisture content was calculated using the following formula:

% Moisture =(Weight of fresh sample − Weight of dried sample) / Weight of fresh sample × 100

Ash Content

Ash content was determined by placing the sample in a pre-weighed crucible and heating it over
a Bunsen burner followed by incineration in a muffle furnace at 550°C until greyish-white ash
was obtained. The crucible was cooled in a desiccator and weighed, and ash percentage was
calculated using standard procedures (AOAC, 2016).

The ash content was calculated using the following formula:

% Ash = Weight of ash / Weight of sample × 100

Crude Fat

Crude fat content was determined using the Soxhlet extraction method. The dried sample was
placed in a thimble and extracted with petroleum ether (40–60°C) for 6–8 hours. After
extraction, the solvent was evaporated, and the residue was dried in a hot air oven, cooled, and
weighed to determine fat content (AOAC, 2016).

% Fat = (Weight of sample − Weight of fat-free sample) / Weight of sample × 100

Crude Fiber

Crude fiber was determined by acid and alkali digestion method. The fat-free sample was first
boiled with 1.25% sulfuric acid, filtered, and washed, followed by boiling with 1.25% sodium
hydroxide. The residue was dried, incinerated, and the loss in weight was used to calculate crude
fiber content (AOAC, 2016).

% Fiber = Weight loss on ignition / Weight of sample × 100

Crude Protein Content

Crude protein content was determined using the Kjeldahl method. The sample was digested with
concentrated sulfuric acid in the presence of a catalyst, followed by distillation and titration of

3
released ammonia. The nitrogen content obtained was multiplied by 6.25 to calculate protein
content (AOAC, 2016).

% Protein = Nitrogen (%) × 6.25

Nitrogen-Free Extract (NFE)

Nitrogen-free extract was calculated by difference using the values obtained from moisture, ash,
protein, fat, and fiber. It represents the carbohydrate content of the herbal tea sample (AOAC,
2016).

% NFE = 100 − (Moisture + Ash + Protein + Fat + Fiber)

Antioxidant Analysis of herbal tea:

Redical scavenging activity using the DPPH method:

The radical scavenging activity of tea extract was determined using the DPPH assay, a widely
used method for evaluating antioxidant potential in herbal infusions. In this method, 1 mL of tea
extract was mixed with 1 mL of freshly prepared DPPH solution (0.1 mM in methanol) and
incubated in the dark at room temperature for 30 minutes to allow the reaction between
antioxidants and free radicals. After incubation, the absorbance was measured at 517 nm using a
UV–visible spectrophotometer, while a control containing only DPPH solution was also
prepared. The decrease in absorbance indicates the ability of tea polyphenols to scavenge free
radicals by donating hydrogen atoms, leading to discoloration of the DPPH solution. Recent
studies have confirmed that tea infusions exhibit significant DPPH radical scavenging activity
due to their high content of phenolic compounds and flavonoids (Gallia et al., 2024).

Formula:

% Inhibition = [(A₀ − A₁) / A₀] × 100

Where:

A₀ = Absorbance of control

A₁ = Absorbance of sample

Total Phenolic Content (TPC) of Tea

4
The total phenolic content of tea extract was determined using the Folin–Ciocalteu method, a
widely accepted assay for estimating phenolic compounds in plant-based beverages. In this
method, 0.5 mL of tea extract was mixed with 2.5 mL of diluted Folin–Ciocalteu reagent and
allowed to react for a few minutes, followed by the addition of 2 mL of sodium carbonate
solution. The mixture was then incubated in the dark at room temperature for 30–60 minutes to
allow the formation of a blue-colored complex due to the reduction of phosphomolybdic–
phosphotungstic reagents by phenolic compounds. After incubation, the absorbance was
measured at 765 nm using a UV–visible spectrophotometer. A blank containing all reagents
except the sample was also prepared. The phenolic content was calculated using a gallic acid
calibration curve and expressed as gallic acid equivalents (GAE). Recent studies have shown that
tea exhibits high total phenolic content due to the presence of bioactive compounds such as
catechins and flavonoids, which contribute significantly to its antioxidant capacity (Zhao et al.,
2023).

Formula:

C = (c × V) / m

Where:

C = Total phenolic content (mg GAE/g extract)

c = Concentration of gallic acid from calibration curve (mg/mL)

V = Volume of extract (mL)

m = Weight of extract (g)

Sensory evaluation:

The tea samples were evaluated for attributes such as appearance, color, aroma, flavor, taste,
mouthfeel, and overall acceptability. A trained panel assessed the samples using a 9-point
Hedonic Scale, where Extremely-9, Liked very much-8, Liked moderately-7, Liked slightly-6,
Neither liked nor disliked-5, Disliked slightly-4, Disliked moderately-3, Disliked very much-2,
and Disliked extremely-1 (Moreira et al., 2024).

5
Statistical analysis:

The obtained data was statistically analyzed according to analysis of variance (ANOVA)
procedure of statistical package SPSS to determine the level of significance (Zhou et a., 2023)

Reference:

Moreira J, Aryal J, Guidry L, Adhikari A, Chen Y, Sriwattana S & Prinyawiwatkul W (2024).

Tea Quality: An Overview of the Analytical Methods and Sensory Analyses Used in the Most
Recent Studies, Foods, 13(22), 3580

Zhao, Y., Chen, P., Lin, L., et al. (2023). Phenolic composition and antioxidant activity of
different tea extracts. Food Chemistry, 405, 134809.

Gallia, M. C., Ferrari, A., & Bajda, L. (2024). Antioxidant activity and phenolic content of
herbal infusions from medicinal plants. Food Production, Processing and Nutrition, 6, 45.

Official Methods of Analysis of AOAC International

AOAC (2016). Official Methods of Analysis of AOAC International (20th ed.). Association of
Official Analytical Chemists, Washington, DC, USA.

Khan, M. A., et al. (2014). Development and quality evaluation of herbal tea blends. Journal of
Food Processing and Technology, 5(6), 1–5.