Hindawi
Advances in Pharmacological and Pharmaceutical Sciences
Volume 2022, Article ID 7235489, 10 pages
https://doi.org/10.1155/2022/7235489
Research Article
Approaches for the Elimination of Microbial Contaminants from
Lippia multiflora Mold. Leaves Intended for Tea Bagging and
Evaluation of Formulation
Doris Kumadoh ,1,2 Mary-Ann Archer ,1 Michael O. Kyene ,1 Genevieve N. Yeboah,1
Ofosua Adi-Dako ,3 Christina Osei-Asare,4 Emmanuel Adase ,2 Susana Oteng Mintah,5
Hilda Amekyeh ,6 and Alfred A. Appiah7
1Department of Pharmaceutics, Centre for Plant Medicine Research, Mampong-Akuapem, Ghana
2Department of Production, Centre for Plant Medicine Research, Mampong-Akuapem, Ghana
3Department of Pharmaceutics and Microbiology, School of Pharmacy, University of Ghana, Legon, Accra, Ghana
4Department of Pharmaceutics and Microbiology, Central University College, Accra, Ghana
5Department of Microbiology, Centre for Plant Medicine Research, Mampong-Akuapem, Ghana
6Department of Pharmaceutics, School of Pharmacy, University of Health and Allied Sciences, Ho, Ghana
7Department of Phytochemistry, Centre for Plant Medicine Research, Mampong-Akuapem, Ghana
Correspondence should be addressed to Mary-Ann Archer; maryannarcher16@yahoo.com
Received 20 October 2021; Revised 3 February 2022; Accepted 10 February 2022; Published 27 February 2022
Academic Editor: Mounir Tilaoui
Copyright © 2022 Doris Kumadoh et al. )is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Elimination of microorganisms from herbal products has been a major concern due to its implicated health risk to consumers.
Drying of herbal materials has been employed for centuries to reduce the risk of contamination and spoilage. )e present study
adopted three drying approaches in an attempt to eliminate microorganisms from Lippia multiflora tea bag formulation. )is
study also evaluated the tea bags and optimized the extraction procedure. )e L. multiflora leaves for tea bagging were air-dried
and milled (A), oven-dried and milled (B), and microwaved (the milled air-dried leaves) (C). )e moisture contents were
determined at 105°C± 2°C for 2 hours to constant weight. Phytochemical parameters such as phytochemical constituents, total
water extractive, and pH were assessed. )e microbial safety and quality of the L. multiflora tea bags were evaluated using the
British Pharmacopoeia 2019 specifications.)e uniformity of the mass of the formulated tea bags was also determined. Extraction
from the Lippia tea bags was optimized. )e results showed that using the approaches (A, B, and C) adopted for drying and
processing, the moisture contents of the formulated tea bags were in the range of 9.75–10.71% w/w. All the formulated tea bags
contained reducing sugars, phenolic compounds, polyuronides, flavonoids, anthracenosides, alkaloids, saponins, and phytos-
terols.)e pH range of the formulations was 7.11–7.54, whereas the total water extractive values were in the range of 19.12–20.41%
w/w. )e one-way analysis of variance demonstrated no significant difference in the data obtained from the results from A, B, and
C. )e formulation from A was found to be unsafe for consumption due to unacceptable microbial contamination limits.
Microbial load of the formulations from B and C were within the BP specifications. All the batches of the formulations passed the
uniformity of mass test. An optimized extraction procedure was obtained when one tea bag was extracted in 250mL of hot water
within the specified time. L. multiflora leaves meant for tea bagging should be oven-dried or microwaved before tea bagging for
safe consumption.
2 Advances in Pharmacological and Pharmaceutical Sciences
1. Introduction the elimination of microbial contaminants from herbal tea
without adversely affecting the phytochemical constituents
West Africa is endowed with many stouts, woody and ar- and quality properties of the tea. An attempt has also been
omatic perennial shrubs, of which Lippia multiflora Mol- made to do an optimization of the extraction process for the
denke is a typical example. L. multiflora belongs to the formulated tea bags.
Verbenaceae family and grows up to a height of 2.8–4m [1].
L. multiflora is normally found in the forest savanna, sub-
savanna, and transitional areas of Ghana. )e leaves of 2. Materials and Methods
L. multiflora have been formulated into a herbal tea that is 2.1. Plant Material Collection. Fresh leaves of L. multiflora
used because of its aromatic odor and therapeutic property were harvested from the Centre for Plant Medicine Research
of stress relief [2, 3]. )e leaves of L. multiflora are normally (CPMR) farm at Mampong-Akuapem, Ghana (5°55′ 06.6″
used for the management of mild hypertension and as a N, 0° 07′57′57 W) and authenticated by the Plant Devel-
diuretic [4, 5]. Studies on the anti-inflammatory, antima- opment Department, CPMR. )e plant specimen has been
larial, antioxidant [6], analgesic, and antipyretic activities of placed in its herbarium with the identification code CPMR
the L. multiflora have been conducted justifying its tradi- 5073. )e methods that were used for processing the ma-
tional use for the related conditions [4]. )e determination terials before tea bagging have been detailed below.
of the shelf life of L. multiflora leaves (Lippia tea) indicated a
period of 62.97 months as the shelf life in a study conducted
by Kumadoh and colleagues when the product was stored at 2.2. PlantMaterial Processing. )ree different methods were
specified temperatures of 30°C± 2°C/70%± 5% RH (relative used for the processing of the plant material. For each
humidity) [7]. A study on the safety of a powder prepared method, four different batches of tea bags were formulated
from the dried leaves of L. multiflora has also been reported for subsequent analyses. )e tea bags manufactured were
by Djengue and colleagues [8]. Results obtained from this batch coded as LTB1, LTB2, LTB3, and LTB4, respectively.
study showed that the powder displayed microbial safety at
the beginning of storage until at least 18 months of storage
without risk of contamination for the consumer. However, 2.2.1. Methods of Processing: Air-Drying, Oven-Drying, and
powders bought from the markets could not be stored for Microwaving of L. multiflora Mold. Leaves. )e method of
more than 12 months due to pathogenic and flora con- processing the fresh leaves of L. multiflora was modified
taminations [8]. fromMahanom et al [19], Roshanak et al. [20], andManzano
)e presence of microbial contaminants in some herbal Santana et al. [21]. )e fresh leaves of L. multiflora were
products may pose a public health risk to consumers [9, 10] washed in 70% (v/v) ethanol and subsequently washed with
with concerns about microbial contamination in herbal water to remove all residual ethanol, dust, and unwanted
products being a major reason why some consumers avoid materials accumulated on the leaves. )e leaves were air-
herbal products in favor of orthodox medicines [11]. When dried for 7 days on wooden-structured shelves with their
plant material processing strategies such as harvesting, bottoms covered with a wire mesh in a well-ventilated drying
drying, milling, tea bagging, packaging, storage, and dis- room for the air-drying method. In the oven-drying method,
tribution are not properly done by following good the leaves, after being washed, were dried in an oven (IGNIS,
manufacturing practices, microbial contamination may re- Italy) at a temperature of 70°C for 2 hours. Dried leaves
sult [12, 13]. Some of the ways by which microbial con- obtained using the two methods were then milled to obtain a
tamination of food products can be eliminated include coarse powder (ca 2mm2 in size) using a stainless-steel
efficient drying, chilling, curing, and freezing [14]. Irradi- hammer mill (JICA, Japan) machine and sieved using a
ation, sterilization, and pasteurization could also make 2mm mesh sieve. )e third method of processing involved
microorganisms inactive for growth and multiplication, microwaving of a sample of the milled plant material ob-
thereby preventing contamination. In addition, steam tained from the air-drying method for 5 minutes. Tea
sterilization and ozonation have been reported to be useful bagging of the material was done using a tea-bagging ma-
in controlling microbial growth in herbs [15]. Studies have chine (GATBPM, India). )e obtained tea bags were
shown L. multiflora microbial contamination could be re- packaged in ziplock bags and placed in a box. )e procedure
duced by steam treatment [16]. Some factors that should be was repeated for four different batches of the product for
considered when selecting methods for microbial decon- each processing method.
tamination include safety, quality, efficacy, practicability,
and cost. Care must be taken to prevent the deterioration of
essential constituents such as antioxidants and other phy- 2.3. Determination of Moisture Content of Powder for Tea
tochemical compounds in plant materials during the mi- Bagging. )emoisture contents (percentage) of 3 g of milled
crobial decontamination process [16]. Microorganisms material obtained from each processing method which were
contaminate various parts of herbal plants, including leaves, to be used in the preparation of the tea bags were determined
stems, flowers, seeds, and roots [17, 18]. It is important to using a moisture content analyzer (Kern, MLB 50-3, Ger-
eliminate microbial contaminants in the herbal tea of many) at a temperature of 105°C± 2°C for 2 h to constant
L. multiflora in order to ensure its safety and quality.)e aim weight. )e moisture content was deduced as the percentage
of this study is to develop a simple and practical method for of the lost weight divided by the initial weight. )is was
Advances in Pharmacological and Pharmaceutical Sciences 3
determined in triplicate for each method and the mean bacterial counts and malt extract agar for fungi
percentage moisture content calculated [22]. identification.
)e media were prepared according to the manufac-
turer’s instructions and incubated at 37°C for 24 h for
2.4. Phytochemical Analysis. )e formulated tea bags were bacterial screening and at 25°C for 5 days for fungal
assessed for phytochemical parameters such as phyto- screening. At the end of the incubation period, the number
chemical constituents, total water extractive, and pH. of colony-forming units per gram (CFU/g) was calculated by
Standard phytochemical screening tests were used to detect multiplying the average number of colonies by the dilution
secondary metabolites from L. multiflora. Mayer’s tests for factor. All microbial analyses were carried out in triplicate.
alkaloids, Fehling’s test for free reducing sugars, the Lie- For investigating the presence of pathogenic bacteria, se-
bermann–Burchard test for phytosterol and triterpenes, lective media used for the identification of Escherichia coli,
froth test for saponins, Shinoda’s test for flavonoids, Staphylococcus spp., and Salmonella spp. were MacConkey
Borntrager’s test for free anthracenosides, and ferric chloride agar, mannitol salt agar, and xylose lysine deoxycholate agar,
solution test for phenolics were used [23]. respectively.
2.5. Determination of pH. A pH meter was calibrated with 2.8.MassUniformityof theFormulatedTeaBags. )e average
standard pH buffer solutions beginning with buffers of pH 7 mass of 20 randomly selected units of the tea bags was
and 10 (Riedel-de Haen, Germany) followed by that with pH determined by weighing a single filled tea bag, opening it
4 (Reagacon, Ireland) to determine the linearity of response whilst making sure no fragments were lost with complete
of the electrode. To determine the pH, a cup was filled with a emptying. )e weight of the empty bag was noted and the
portion of a 1%w/v infusion prepared from the tea bags to weight of the content was calculated by deduction. )e
obtain a preliminary value. Subsequent readings were taken procedure was repeated on the 19 remaining tea bags. )e
on additional portions of the same sample to yield more uniformity of mass was then determined. )is procedure
constant pH values that were reproducible to ±0.04 units and was done in triplicate for all four batches of products ob-
that showed drifts of less than ±0.04 unit in 2min, and the tained from the three different processing methods [22].
maximum pH value obtained recorded. )e procedure was
done in triplicate for all four batches of products from the
three processing methods [24]. 2.9. Extraction Method Optimization. )e method for op-timization of the extraction was modified from Chandini
et al. [26] and Yuto et al. [27]. One tea bag (2 g net weight)
2.6. Determination of Total Water Extractive. One gram of was soaked in 300mL of hot water (freshly boiled), allowed
the material in a Lippia tea bag was weighed into a 250mL to extract for about 10 minutes, and filtered. )e volume of
flask with a stopper. Next, 100mL of distilled water was the filtrate was recorded and the total solid residue was
added and the mixture was placed on a water bath for 1 hour. determined.)e total extract available in the filtrate was then
)e extract was filtered using Johnson test paper (Qualitative calculated. )e procedure was repeated using various vol-
filter paper, grade 304; 125mm) and 10mL of the filtrate was umes of the hot water (250, 200, and 150mL) for the ex-
pipetted for drying in an evaporating dish at 105°C for 1 h to traction procedure. )e procedure was done in triplicate for
a constant weight. )e evaporating dish containing the each extraction volume. )e same procedure above was also
residue was transferred into a desiccator to cool and later repeated using two tea bags (4 g net weight).
weighed [25].
)e weight of the residue was calculated as follows: 2.10. Statistical Analyses. )e standard deviations and
Weight of residue� (weight of dish + residue)− (weight of means of the replicate determinations were analyzed using
empty dish). Microsoft excel, 2016 version. )e one-way analysis of
)e total water extractive value was calculated as follows: variance (ANOVA) was used to analyze the pH, moisture
percentage weight per weight content, and total water extractive data obtained from using
w weight of residue the three different processing methods. GraphPad Prism
􏼒% 􏼓 � × 100. (1)
w initial weight in 10mL version 7 was used to prepare all the plots. In the optimi-
zation of extraction study, linear regression analyses were
employed.
2.7. Microbial Load Test Using Total Bacterial, Fungal, and 3. Results and Discussion
Pathogenic Bacterial Counts. )e microbial safety and
quality of the L. multiflora tea bags were assessed using the 3.1. Physicochemical Properties and Phytoconstituents Present
British Pharmacopoeia 2019 specifications. )e tests were in the Formulated Tea Bags. )e results from the analyses of
used to quantify the number of bacteria and fungi isolated the four different batches of tea bags obtained using the three
that are able to grow aerobically in 1 g of sample. All mi- processing methods as presented in Table 1 showed that all
crobial analyses were carried out in triplicate. Viability was samples produced by the three processing methods con-
assessed by the pour plate method using plate count agar for tained phytochemical constituents such as reducing sugars,
4 Advances in Pharmacological and Pharmaceutical Sciences
Table 1: Phytochemical properties of the formulated tea bags.
Processing approach
Phytochemical test Inference A B C
LTBI LTB2 LTB3 LTB4 LTBI LTB2 LTB3 LTB4 LTBI LTB2 LTB3 LTB4
Reducing sugar Brick-red colouration + + + + + + + + + + + +
Saponins Formation of froth whichpersists for 10min + + + + + + + + + + + +
Phenolic
compounds Dark green colouration + + + + + + + + + + + +
Polyuronides Violet precipitation + + + + + + + + + + + +
Flavonoids Orange colouration + + + + + + + + + + + +
Anthracenosides Red colouration + + + + + + + + + + + +
Alkaloids Cream precipitation + + + + + + + + + + + +
Phytosterols Dark green colouration + + + + + + + + + + + +
Approach A: air-drying of the fresh leaves of L. multifloraMold. before tea bagging; Approach B: oven-drying of the fresh leaves of L. multifloraMold. before
tea bagging; Approach C: Microwaving of air-dried processed sample of L. multiflora Mold. before tea bagging. +means present.
phenolic compounds, flavonoids, alkaloids, saponins, pol- methods showed no significant difference (P> 0.05) in the
yuronides, anthracenosides, and phytosterols. )e presence means of the extractive values obtained, as has been indi-
of these phytochemicals has been reported [1, 28] where the cated in Figure 1. )is indicates that the different processing
parameters were not affected by the drying methods used. A techniques did not affect the total water extractives.
similar study was conducted by Manzano Santana et al. [21] According to a study on the determination of total water
where the drying methods used on Ilex guayusa leaves did extractives of poly-herbal formulations, the water-soluble
not affect the phytochemical constituents present in the extractives of the tested materials ranged from 8.23 to
plant. It did, however, increase the concentration of sec- 34.52% [40]. From the results obtained as indicated in
ondary metabolites in the aqueous and ethanol extracts Figure 1, the total water extractive obtained was in the range
tested. Only alkaloids were found in the leaves of of 19.53 to 20.93%, which indicates a good extraction for all
L. multiflora collected from six districts in Benin, according three drying methods. A lower total extractive value may
to Djengue et al. [29]. )e current study validates the indicate an exhausted material, contamination, improper
previous studies reported. )e occurrence of these essential drying, storage, or preparation, whereas a higher total ex-
phytochemical constituents in L. multiflora may be con- tractive value may also indicate the presence of more water-
tributed to the antifungal, antibacterial, antiedema, anti- soluble contents in the plant material [40].
inflammatory, antiviral, antimalarial, and anti-stress activ- )e results (Figure 2) illustrated that the pH range of the
ities of the plant [28–33]. )e results obtained indicate the products was 3.00 to 5.09. Statistical analysis of this result
oven temperature of 70°C did not affect the presence of the using the one-way ANOVAmethod of the samples prepared
phytochemical constituents determined in the samples. )e showed no significant difference (P> 0.05) in the pH values
reduction in the amounts of volatile compounds in plant recorded for the different batches prepared via the different
materials during oven-drying depends on the volatility and processing methods. )is indicates that the processing
chemical structure of the constituents [34]. Radünz et al. [35] methods did not affect the pH of the products. In a previous
observed no significant difference in the essential oil content reported research project [41], the pH of some herbal
of fresh leaves of L. sidoides compared to the leaves that were medicines was determined to ascertain their acidity or al-
oven-dried at temperatures of 40°C to 70°C. Meanwhile, the kalinity. Additionally, Onwordi et al. [42] have reported a
essential oil content of the sample dried in ambient air was pH range of 3.35–8.00 for some liquid herbal medicines sold
8% lower. When this evaluation was repeated, similar results in Nigeria, whereas an acidic pH range of 1.05–3.55 has also
were obtained with no significant qualitative changes in been reported for liquid herbal medicinal products [43].
essential oils (thymol) comparable to the fresh plant [36]. Meanwhile, the obtained pH falls within the reported tol-
L. aliba leaves were examined under six different drying erable pH for natural plants, i.e., 4.0–7.5 [44]. Generally, the
treatments, using air at ambient temperature and air heated buffer systems of the body neutralize the accumulated acids
up to 80°C. )e investigators concluded that drying of the and when this system is burdened, alkaline minerals from
plant for marketing purposes can be carried out using air bones and vital organs are used by the body to neutralize
that is heated to 40−80°C [37]. However, the changes in the acidic compounds. )is results in the deterioration of the
chemical composition of essential oils and compounds bones and the organs, leading to osteoporosis and unde-
present in herbal tea/products depend on individual me- sirable effects on the immune, circulatory, digestive, respi-
dicinal plants, the drying temperature conditions, and the ratory, and other body systems [45]. )e ingestion of herbal
drying time [38, 39]. medicines with high acidic pH reduces the pH of the fluids in
)e one-way ANOVA results for the total water ex- the stomach above the acceptable level required for meta-
tractives (means, standard deviation (SD) of the four dif- bolism, thus hindering enzyme action [45]. Enzymes are
ferent batches) after using the three different processing sensitive to pH changes and are known to function best at a
Advances in Pharmacological and Pharmaceutical Sciences 5
25 15 **
20 ns
15 10
10
5
5
0
A B C 0
Processing Approach A B C
Processing Approach
Figure 1: Graph of mean total water extractive from the various
processing approaches of the formulated tea bags. Approach A: air- Figure 3: Graph of the mean moisture content. Approach A: air-
drying of the fresh leaves of L. multifloraMold. before tea bagging; drying of the fresh leaves of L. multifloraMold. before tea bagging;
Approach B: oven-drying of the fresh leaves of L. multiflora Mold. Approach B: oven-drying of the fresh leaves of L. multiflora Mold.
before tea bagging; Approach C: microwaving of air-dried pro- before tea bagging; Approach C: microwaving of air-dried pro-
cessed sample of L. multifloraMold. before tea bagging. Values are cessed sample of L. multifloraMold. before tea bagging. Values are
mean± SD. mean± SD. P> 0.05 not significant (ns), P< 0.01 significant (
∗∗).
prevention of fungal and bacterial growth. Eko et al. [46] in a
10 moisture content determination study reported a low
8 moisture content of 8.6% for the flowers, roots, leaves, fruits,
and bark of medicinal plants that had been oven-dried, while
6 the moisture content of air-dried samples was 9.81%. Ahmad
4 et al. [47] have also reported moisture contents of 11.0% and
12.14% for field-grown leaves and roots, respectively, of
2 Rauvolfia serpentia at 105°C. )e moisture content of herbal
plants depends on the nature and type of herb, postharvest
0
A B C practices, the age of the plants, and the chemical compo-
Processing approach sition of the plant. )e moisture contents of L. multiflora
Figure 2: Graph of mean pH values obtained. Approach A: air- leaf, flower, and market powders, as evaluated by Djengue
drying of the fresh leaves of L. multifloraMold. before tea bagging; and his team [8], were found to be 7.42%, 9.89%, and
Approach B: oven-drying of the fresh leaves of L. multiflora Mold. 11.67%, respectively.)e results of the present study showed
before tea bagging; Approach C: microwaving of air-dried pro- that the moisture content of the L. multiflora leaves was in
cessed sample of L. multifloraMold. before tea bagging. Values are the range of 9.75–10.71%, which is consistent with the re-
mean± SD. ported results [8].
specific pH range [45]. However, the pH values obtained for 3.2. Uniformity of Mass of the Formulated Tea Bags. For the
the Lippia tea bags from the three processing methods (A, B, uniformity of mass, the acceptance criteria for weights be-
and C) in this study ranged from 7.11 to 7.54, which is within tween 1.5 g and 2 g is that not more than two sachets should
the acceptable range of 4.0–7.5 reported by Edebi and deviate by more than 10% from the average mass and none
Gideon [44]. )is indicates that the teas produced from the should deviate by more than 20% [22]. As can be observed
various processing methods may be safe for consumption from Table 2 the batches passed the uniformity of mass test.
with regard to their pH. It can be inferred that the tea-bagging machine helped
)emoisture content of the air-dried sample (A) was the ensure the uniform filling of tea bags. In addition, it helps to
highest, with an average value of 10.71%, followed by that of provide a product that is convenient for patient usage
the oven-dried sample (B, 9.83%). )e microwave-dried compared to powders that have not been bagged.
sample (C) had a moisture content of 9.75%. Nonetheless,
statistical analysis using one-way ANOVA demonstrated no
significant (P ˂ 0.01) difference between the recorded values 3.3. Microbial Load of L. multiflora Mold. Tea Bags.
for A compared to B and C as shown in Figure 3. )is Results of the microbial load analyses of the air-dried
indicates that the processing methods affected the moisture L. multiflora Mold. tea bags have been presented in Table 3.
contents of the products. A higher moisture content as seen It can be observed that all the four batches of the products
in the air-dried sample may lead to increased susceptibility failed the microbial load test.)e quality and safety of herbal
to microbial growth. Moisture content can be understood as teas for commercial use can be promoted if microbial
the amount of water contained in a material or substance. contamination levels are addressed through appropriate
)e moisture content of a herbal plant helps to estimate the quality control processes during the preparation of the plant
total solid matter and it is one of the major factors re- material for tea bagging. )ough some consumers believe
sponsible for the deterioration of solid, powdered herbal that hot water steeping of herbal teas could reduce microbial
drugs and formulations. A relatively lower moisture content contamination, once there is a high level of contamination
is always desirable for higher drug stability and the beyond acceptable limits, steeping of herbal tea in hot or
Mean pH Mean total water 
extractive (% w/w)
Mean moisture
content (% w/w)
6 Advances in Pharmacological and Pharmaceutical Sciences
Table 2: Uniformity of mass of the formulated tea bags.
Processing Batch of Number of tea bags deviating by more than 10% Number of tea bags deviating by more than 20%
approach product from mean (N� 20) from mean (N� 20)
LTB1 2 0
A LTB2 1 0LTB3 2 0
LTB4 2 0
LTB1 1 0
B LTB2 2 0LTB3 1 0
LTB4 1 0
LTB1 1 0
C LTB2 0 0LTB3 1 0
LTB4 2 0
Approach A: air-drying of the fresh leaves of L. multifloraMold. before tea bagging; Approach B: oven-drying of the fresh leaves of L. multifloraMold. before
tea bagging; Approach C: microwaving of air-dried processed sample of L. multiflora Mold. before tea bagging.
Table 3: Microbial load analyses of tea bags prepared from the air-dried leaves of L. multiflora Mold.
Test conducted LTB1 (cfu/g) LTB2 (cfu/g) LTB3 (cfu/g) LTB4 (cfu/g) Acceptance criterion (BP, 2019)
1TAMC/37 °C/24 hours/PCA 8.6×108 TNTC TNTC 7.3×109 ≤5.0×107
2TYMC/25 °C/5 days/MEA 4.9×107 TNTC TNTC 5.1× 106 ≤5.0×105
Escherichia coli (E. coli) + − + − Absence
Salmonella spp. − − − − Absence
Staphylococcus spp. − + − + Absence
BP, British Pharmacopoeia; 1TAMC: total aerobic microbial counts, 2TYMC: total yeast and mold counts; TNTC: too numerous to count; −: absent; +:
present; PCA: plate count agar; MEA: malt extract agar.
boiling water alone may not be able to clear the contami- From Table 4, it can be observed that the total aerobic
nation to acceptable levels [48]. Drying plays a pivotal role in bacteria, yeast, and mold counts in all the four batches of
good manufacturing practices during the production of oven-dried L. multiflora Mold. leaves before tea bagging
herbal products, as it helps reduce the moisture content of were within the acceptable specification limits, which could
materials, thus preventing microbial contamination and be due to the drying conditions employed. Kulshrestha
prolonging the shelf life of the materials [49]. )e presence et al. [55] have reported that drying herbs at a relatively
of aerobic bacteria and fungi beyond acceptable limits may high temperature decreases the total microbial counts.
be due to frequent multiplication in the air under ambient Research has also revealed that L. multiflora leaves contain
temperature conditions and microbial contaminants settling some essential oils that have antimicrobial properties [13].
on leaves during the drying process [50]. E. coli was present It has been revealed in some studies that essential oils in the
in the LTB1 and LTB3 samples. )e presence of E. coli and leaves of plants have a strong inhibitory effect on the
Staphylococcus spp. has been reported in some homemade growth of Staphylococcus aureus and Enterococcus hirae
and commercial herbal medicine [51] as well as in some and a moderate inhibitory effect on Candida albicans and
dried herbs and teas [52]. E. coli may cause urinary tract Saccharomyces cerevisiae [30, 56, 57]. Specially, essential
infections, pneumonia, and diarrhea [53]. Contamination oils such as carvacrol and thymol have been shown to have
from Staphylococcus spp. could also cause cellulitis, stom- antimicrobial properties in previous studies [32, 58, 59].
achache, scalded-skin syndrome, and impetigo [54]. )e )is may account for the low microbial contamination
relatively higher moisture content of the air-dried powders observed in L. multiflora tea bag samples investigated using
(Figure 3) possibly resulted in the failing of the microbial oven-dried and microwave-dried methods. Meanwhile,
load test. )e high moisture content made the samples more high microbial contamination was observed in the air-dried
susceptible to microbial contamination in addition to samples.
contamination from the environment during the air-drying It can be observed from Table 5 that tea bags produced
process. A study by Djengue et al. [8] showed that high from the microwaved air-dried samples passed the mi-
moisture and ash contents in the powders of L. multiflora crobial load tests. In comparison to the air-dried sample,
favor the growth and development of yeast and molds. )e which failed the tests, it can be deduced that microwaving
load detection for Salmonella spp. in this study was within of the samples may have caused deactivation of the mi-
the acceptable criteria. Similar results have also been re- crobial agents present in the air-dried sample. Microwaves
ported by Djengue and his team [8], who found the coliform use nonionizing electromagnetic waves of 1m to 1mm and
detection threshold for Salmonella spp. in dried L. multiflora frequencies between 0.3 and 300GHz [60]. )e application
leaves to be below the acceptable limits. of microwave treatment on spices and herb samples has
Advances in Pharmacological and Pharmaceutical Sciences 7
Table 4: Microbial load analyses of tea bags prepared from the oven-dried leaves of L. multiflora Mold.
Test conducted LTB1 (cfu/g) LTB2 (cfu/g) LTB3 (cfu/g) LTB4 (cfu/g) Acceptance criterion (BP, 2019)
1TAMC/37°C/24 h/PCA 1.2×103 1.1× 103 1.8×102 2.5×104 ≤5.0×107
2TYMC/25°C/5 days/MEA 6.0×102 5.5×102 3.9×103 7.4×104 ≤5.0×105
E. coli − − − − Absence
Salmonella spp. − − − − Absence
Staphylococcus spp. − − − − Absence
BP, British Pharmacopoeia; 1TAMC: total aerobic microbial counts; 2TYMC: total yeast and mold counts; −: absent; +: present; PCA: plate count agar; MEA:
malt extract agar.
Table 5: Microbial load analyses of tea bags prepared from the microwaved air-dried leaves of L. multiflora Mold.
Test conducted LTB1 (cfu/g) LTB2 (cfu/g) LTB3 (cfu/g) LTB4 (cfu/g) Acceptance criterion (BP, 2019)
1TAMC/37°C/24 h/PCA 8.7×104 1.3×103 2.6×104 6.9×105 ≤5.0×107
2TYMC/25°C/5 days/MEA 6.5×103 9.1× 104 4.3×103 3.8×103 ≤5.0×105
E. coli − − − − Absence
Salmonella spp. − − − − Absence
Staphylococcus spp. − − − − Absence
BP, British Pharmacopoeia; 1TAMC: total aerobic microbial counts; 2TYMC: total yeast and molds counts; −: absent; +: present; PCA: plate count agar; MEA:
malt extract agar.
0.8
0.6
0.4
0.2
0.0
1 2 1 2 1 2 1 2
Number of Tea bags used
150 mL 250 mL
200 mL 300 mL
Figure 4: Weight of extracts obtained from the tea bags in the extraction procedure. “1” indicates one tea bag (2 g) and “2” indicates two tea
bags (4 g).
Table 6: Results of optimization of extraction from Lippia tea bags.
Weight of tea bags used in Volume of water used in Volume of filtrate obtained Total solid residue Total extract
extraction (g) extraction (mL) (mL) (% w/v) inN� 3 N� 3 filtrate (g)
2 300 265± 5 0.15± 0.02 0.400± 0.05
2 250 215± 2 0 .25± 0.01 0.538± 0.02
2 200 170± 5 0.25± 0.02 0.425± 0.03
2 150 130± 2 0.20± 0.04 0.26± 0.05
4 300 275± 4 0.10± 0.01 0.275± 0.03
4 250 225± 3 0.30± 0.01 0.675± 0.02
4 200 180± 5 0.25± 0.02 0.450± 0.04
4 150 120± 2 0.55± 0.04 0.660± 0.05
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Total weight of
extract from extraction (g)
8 Advances in Pharmacological and Pharmaceutical Sciences
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