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Citation: Benjamin, M.A.Z.; Ng, S.Y.;
Saikim, F.H.; Rusdi, N.A. The Effects
of Drying Techniques on
Phytochemical Contents and
Biological Activities on Selected
Bamboo Leaves. Molecules 2022,27,
6458. https://doi.org/10.3390/
molecules27196458
Academic Editor: Randy Purves
Received: 26 August 2022
Accepted: 24 September 2022
Published: 30 September 2022
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Licensee MDPI, Basel, Switzerland.
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Attribution (CC BY) license (https://
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4.0/).
molecules
Article
The Effects of Drying Techniques on Phytochemical Contents
and Biological Activities on Selected Bamboo Leaves
Mohammad Amil Zulhilmi Benjamin , Shean Yeaw Ng , Fiffy Hanisdah Saikim and Nor Azizun Rusdi *
Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Jalan UMS,
Kota Kinabalu 88400, Sabah, Malaysia
*Correspondence: azizun@ums.edu.my
Abstract:
The therapeutic potential of bamboos has acquired global attention. Nonetheless, the
biological activities of the plants are rarely considered due to limited available references in Sabah,
Malaysia. Furthermore, the drying technique could significantly affect the retention and degra-
dation of nutrients in bamboos. Consequently, the current study investigated five drying meth-
ods, namely, sun, shade, microwave, oven, and freeze-drying, of the leaves of six bamboo species,
Bambusa multiplex
,
Bambusa tuldoides
,Bambusa vulgaris,Dinochloa sublaevigata,Gigantochloa levis, and
Schizostachyum brachycladum
. The infused bamboo leaves extracts were analysed for their total phe-
nolic content (TPC) and total flavonoid content (TFC). The antioxidant activities of the samples were
determined via the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2
0
-azino-bis(3-ethylbenzothiazoline-6-
sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays, whereas their toxicities
were evaluated through the brine shrimp lethality assay (BSLA). The chemical constituents of the sam-
ples were determined using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The
freeze-drying method exhibited the highest phytochemical contents and antioxidant activity yield,
excluding the B. vulgaris sample, in which the microwave-dried sample recorded the most antioxidant
and phytochemical levels. The TPC and TFC results were within the
2.69 ±0.01–12.59 ±0.09 mg
gallic acid equivalent (GAE)/g and 0.77
±
0.01–2.12
±
0.01 mg quercetin equivalent (QE)/g ranges, respec-
tively. The DPPH and ABTS IC
50
(half-maximal inhibitory concentration) were 2.92
±
0.01
–4.73 ±0.02 and
1.89–0.01 to 3.47
±
0.00
µ
g/mL, respectively, indicating high radical scavenging activities. The FRAP
values differed significantly between the drying methods, within the 6.40
±
0.12–36.65
±
0.09 mg Trolox
equivalent (TE)/g range. The phytochemicalcontents and antioxidant capacities exhibited a moderate
correlation, revealing that the TPC and TFC were slightly responsible for the antioxidant activities. The
toxicity assessment of the bamboo extracts in the current study demonstrated no toxicity against the
BSLA based on the LC
50
(lethal concentration 50) analysis at >1000
µ
g/mL. LC-MS analysis showed that
alkaloid and pharmaceutical compounds influence antioxidant activities, as found in previous studies.
The acquired information might aid in the development of bamboo leaves as functional food items, such
as bamboo tea. They could also be investigated for their medicinal ingredients that can be used in the
discovery of potential drugs.
Keywords:
drying methods; antioxidant activities; brine shrimp lethality assay; phytochemical
contents; bamboo leaves
1. Introduction
Drying is a crucial stage during post-harvest because it aids in preventing enzymatic
breakdown and microbial development while retaining the beneficial characteristics of the
dried plants [
1
]. Plant leaves are dried either naturally or via artificial methods. Conven-
tional techniques, such as open sun- and shade-drying at ambient temperatures, are still
employed in rural regions [
1
]. Nevertheless, due to the uncontrollable conditions of the
methods, guaranteeing the safety, efficacy, and consistency of the dried products represents
a challenge [
2
]. Currently, conventional air oven-drying is a standard technique to dry
Molecules 2022,27, 6458. https://doi.org/10.3390/molecules27196458 https://www.mdpi.com/journal/molecules
Molecules 2022,27, 6458 2 of 23
food, although it frequently alters the nutritional value, flavour, and texture of the food and
might oxidise and degrade heat-sensitive polyphenols [
3
,
4
]. Alternatively, various artificial
drying methods, including microwave and freeze-drying, have been utilised to rapidly dry
substantial amounts of leaves with adequate quality [
5
]. Hence, fresh bamboo has a high
moisture content and needs drying to avoid microbial damage and mould, making it ready
for further processing, storage, transportation, and utilisation [6].
The nutritive and therapeutic potential of bamboo leaf extracts in the food and pharma-
ceutical industries have garnered attention worldwide [
7
]. Biologically, bamboo leaves are
rich in polyphenols, flavonoids, and other secondary plant metabolites [
8
,
9
]. The plants are
also widely utilised in traditional Asian medicine to treat arteriosclerosis, cardiovascular
disease, hypertension, certain cancers, oedema, diarrhoea, vomiting, extreme thirst, and to
improve the flavour and colour of foods [
8
,
9
]. Moreover, bamboos are natural antioxidants
that possess the potential to be utilised as novel food additives in edible oils, fish, and meat
products [10].
For many years, bamboo leaf tea has been considered a delicious and healthy drink
in Asian countries [
11
]. Some edible bamboos, such as Bambusa sp., are consumed as tea
and pickles due to their high nutritional and mineral values [
12
]. Zhucha, an ancient
Uyghur treatment, is produced from bamboo leaves and green tea, which possess superior
effectiveness and lipid-reducing effects [
13
]. Sasa quelpaertensis, a bamboo species endemic
to Jeju Island, South Korea, is ingested as a medicinal tea for its anti-diabetic, diuretic,
and anti-inflammatory properties [
14
]. In Japan, Sasa veitchii (or Kuma-zasa) is widely
employed as an ornamental food trimming and in folk medicines. Furthermore, Kuma-zasa
leaves have been utilised in the medical field to treat burns and urinary hesitancy [15].
Phenolics and flavonoids constituents are responsible for the functional efficacy of
herbs. Drying herbal plants could inhibit bacterial growth, increase sample quality, and pre-
vent the oxidation of their chemical contents. Consequently, the drying technique employed
could considerably affect the degradation of the phytochemical and antioxidant contents of
a plant. In this regard, toxicity studies should be accommodated in parallel with antioxidant
activity to ensure their safe use as functional food materials. Nevertheless, the impacts of
the methods on the quality of bamboo leaves have not been explored in depth. The present
study investigated bamboo leaves and optimised their appropriate drying processes by
evaluating the effects of five different drying methods (sun, shade, microwave, oven, and
freeze-drying) on the phytochemical content and antioxidant activities of different bamboo
species. Moreover, the toxicity effects were determined through a brine shrimp lethality
assay (BSLA) of the six bamboo species selected. Using liquid chromatography–tandem
mass spectrometry (LC-MS/MS), further investigation was performed on the chemical
constituents of six different types of bamboo species.
2. Results and Discussion
2.1. The Phytochemical Contents
2.1.1. Total Phenolic Content
The total phenolic content (TPC) data of the six bamboo species assessed in the cur-
rent study are presented in Table 1. Drying considerably increased the TPC (p< 0.05)
of the extracts, and the increment pattern was the lowest in the sun-dried G. levis, fol-
lowed by
S. brachycladum
,B. vulgaris,B. tuldoides,B. multiplex, and D. sublaevigata. Similar
results were documented by Singhal et al. [
16
], who reported that sun-dried B. vulgaris
shoots recorded the lowest TPC (195.05
±
9.82) compared with the tray- (229.6
±
54.25),
oven- (227.55 ±7.77)
, microwave- (224.95
±
49.05), and freeze- (227.66
±
87.12) dried spec-
imens. The long drying time, which exposed the samples to the atmosphere, resulted in
degradation from the oxidation of the phenolic compounds and might explain the low TPC
of the sun-dried samples [
17
]. Enzymatic reactions might also contribute to the loss of the
phenolic chemicals during conventional drying procedures [16].
Molecules 2022,27, 6458 3 of 23
Table 1. The TPC and TFC of the selected bamboo extracts dried with different methods.
Drying Methods TPC 1TFC 2
B. multiplex
Fresh 35.64 ±0.09 b0.87 ±0.01 f
Sun-drying 5.09 ±0.01 d1.13 ±0.00 c
Shade-drying 5.42 ±0.02 c1.26 ±0.00 b
Microwave-drying 5.44 ±0.05 c1.04 ±0.01 d
Oven-drying 5.18 ±0.00 d0.89 ±0.00 e
Freeze-drying 5.74 ±0.06 a1.62 ±0.01 a
B. tuldoides
Fresh 35.26 ±0.01 d1.65 ±0.01 d
Sun-drying 4.31 ±0.03 f1.17 ±0.00 f
Shade-drying 4.92 ±0.01 e1.41 ±0.00 e
Microwave-drying 5.91 ±0.00 a2.06 ±0.00 b
Oven-drying 5.60 ±0.04 c1.69 ±0.02 c
Freeze-drying 5.84 ±0.01 b2.11 ±0.00 a
B. vulgaris
Fresh 34.64 ±0.05 e0.84 ±0.00 d
Sun-drying 4.24 ±0.00 f0.81 ±0.00 e
Shade-drying 5.45 ±0.00 d0.85 ±0.00 d
Microwave-drying 6.17 ±0.04 a1.10 ±0.01 a
Oven-drying 5.77 ±0.03 b0.94 ±0.01 c
Freeze-drying 5.58 ±0.01 c0.96 ±0.00 b
D. sublaevigata
Fresh 38.26 ±0.05 b0.78 ±0.00 b,c
Sun-drying 7.38 ±0.00 d0.78 ±0.00 b
Shade-drying 8.27 ±0.09 b0.77 ±0.00 c
Microwave-drying 8.20 ±0.02 b0.83 ±0.00 a
Oven-drying 7.91 ±0.01 c0.84 ±0.00 a
Freeze-drying 12.59 ±0.09 a0.84 ±0.00 a
G. levis
Fresh 33.92 ±0.00 d0.81 ±0.01 c
Sun-drying 2.69 ±0.01 f0.77 ±0.01 d
Shade-drying 3.68 ±0.02 e0.84 ±0.00 b
Microwave-drying 4.60 ±0.03 b0.82 ±0.01 c
Oven-drying 4.29 ±0.00 c0.84 ±0.00 b
Freeze-drying 4.78 ±0.01 a0.87 ±0.01 a
S. brachycladum
Fresh 34.34 ±0.09 d1.85 ±0.01 b
Sun-drying 4.23 ±0.01 e0.98 ±0.00 f
Shade-drying 4.34 ±0.04 d1.31 ±0.01 e
Microwave-drying 5.01 ±0.03 c1.70 ±0.00 d
Oven-drying 5.30 ±0.09 b1.74 ±0.00 c
Freeze-drying 5.61 ±0.01 a2.12 ±0.01 a
The values represent the means
±
standard deviations of three replicates. Different letters (within a column)
indicate significant differences (one-way ANOVA, Duncan’s multiple comparison test, p< 0.05).
1
TPC was
expressed as mg gallic acid equivalent to 1 g of dried sample (mg GAE/g).
2
TFC was expressed as the mg
quercetin equivalent to 1 g of dried sample (mg QE/g). 3Fresh sample was expressed as control variable.
The drying techniques employed in the present study were incapable of inactivating
degradative enzymes, such as polyphenol oxidases, which were responsible for degrading
phenolic compounds during lengthy drying periods [
18
]. The stability of phenolic chemicals
in herbal infusions was also reported to be affected by drying temperatures [
19
]. The results
demonstrated that the freeze-dried B. multiplex,D. sublaevigata,G. levis, and S. brachycladum
Molecules 2022,27, 6458 4 of 23
recorded the highest TPC. The findings aligned with a report that recorded low-temperature
drying techniques, including freeze-, vacuum-, and infrared-radiation-drying, enhanced the
retention of bioactive chemicals and antioxidant activities in Dendrobium officinale bamboo
shoots [20].
The influences of harvesting season and drying method on the phenolics, flavonoids,
triterpenoids, and antioxidative activities of the leaves of two bamboo species,
Pleioblastus kongosanensis f. aureostriatus and Shibatea chinensis, were observed [
21
]. The
study also found that freeze-, vacuum-, and microwave-oven-drying procedures resulted
in significantly different outcomes [
21
]. In another investigation, microwave-drying tech-
niques produced the highest quality dried herbs faster than other methods [22]. Similarly,
the B. tuldoides and B. vulgaris specimens evaluated in this study subjected to microwave-
drying documented the highest TPC.
2.1.2. Total Flavonoid Content
Table 1summarises the total flavonoid content (TFC) of the bamboo samples assessed
in the current study. Drying notably (p< 0.05) enhanced the TFC of the bamboo extracts.
The sun-dried B. tuldoides,B. vulgaris,G. levis, and S. brachycladum specimens recorded
the lowest TFC. Although the oven-dried B. multiplex and shade-dried D. sublaevigata
specimens documented the least TFC, most reports suggested that sun-drying might not be
a satisfactory technique for some herbs [
22
]. Although the plants are placed in the shade
out of direct sunlight, shade-drying also utilises solar energy as the heat source, which is
similar to sun-drying. Nonetheless, the approach is disadvantageous because it requires
unusually extended drying durations [
23
], which could promote the development of insects
and moulds in high relative air humidity [24].
The freeze-dried B. multiplex,B. tuldoides,D. sublaevigata,G. levis, and
S. brachycladum
specimens in this study demonstrated the highest TFC, supporting the report by
Soesanto [25]
,
who noted that the freeze-dried extracts of B. vulgaris and G. apus shoots exhibited higher
TFC, TPC, and DPPH than the oven-dried samples. Nonetheless, the microwave-dried
B. vulgaris
in this study recorded the highest TFC at 0.14 mg gallic acid equivalent (GAE)/g,
slightly dissimilar from the freeze-dried extracts that exhibited the second highest TFC.
Singhal et al. [
16
] reported that B. vulgaris shoots recorded the second highest TFC yield
when microwave-dried (371.24
±
17.24) following freeze-drying
(438.29 ±6.39)
, which
were superior compared with tray- (284.87
±
34.95), sun-
(346.86 ±26.15)
, and oven-
(327.01 ±19.19) drying.
2.2. Antioxidant Activities
2.2.1. The 2,2-diphenyl-1-picrylhydrazyl Assay
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay is commonly employed to evaluate
the antioxidant activities of samples because the method is simple and inexpensive, and
requires little operating skill and a simple spectrophotometer [
26
]. The IC
50
(half-maximal
inhibitory concentration) value is widely utilised to assess the antioxidant activities of
the samples and is determined from the concentration of antioxidants required to dimin-
ish the initial DPPH concentration by 50% [
27
]. A smaller IC
50
value indicates better
antioxidant attributes.
The DPPH results of this study are presented in Table 2. The sun-dried bamboo sam-
ples recorded high DPPH IC
50
values (lowest free radical scavenging activity). At the same
time, the freeze-dried B. multiplex,B. tuldoides,D. sublaevigata,G. levis, and S. brachycladum
and microwave-dried B. vulgaris extracts exhibited superior DPPH free radical scaveng-
ing activities. One report suggested that a diminished antioxidant content was mainly
attributed to oxidation processes or thermal degradation [
28
]. Consequently, several inves-
tigations have suggested that freeze-drying is preferable in conserving antioxidants [
28
,
29
].
Molecules 2022,27, 6458 5 of 23
Table 2.
The DPPH, ABTS, and FRAP assay results of the selected bamboo extracts dried with
different methods.
Drying Methods DPPH 1ABTS 2FRAP 3
B. multiplex
Fresh 43.73 ±0.00 e2.93 ±0.01 e31.33 ±0.05 d
Sun-drying 4.09 ±0.00 f2.77 ±0.01 d22.39 ±0.11 f
Shade-drying 3.42 ±0.00 b2.59 ±0.01 b29.73 ±0.07 e
Microwave-drying 3.67 ±0.01 d2.72 ±0.01 c33.83 ±0.10 b
Oven-drying 3.49 ±0.00 c2.50 ±0.01 a32.06 ±0.07 c
Freeze-drying 3.20 ±0.00 a2.73 ±0.00 c36.65 ±0.09 a
B. tuldoides
Fresh 43.54 ±0.02 d3.07 ±0.01 f27.68 ±0.12 d
Sun-drying 4.01 ±0.00 f2.70 ±0.00 e19.40 ±0.11 f
Shade-drying 3.82 ±0.01 e2.60 ±0.01 d25.87 ±0.10 e
Microwave-drying 3.37 ±0.01 c2.29 ±0.01 c35.98 ±0.06 a
Oven-drying 3.00 ±0.02 b1.89 ±0.01 a33.83 ±0.01 c
Freeze-drying 2.92 ±0.01 a1.98 ±0.00 b35.83 ±0.05 b
B. vulgaris
Fresh 43.59 ±0.01 c2.74 ±0.00 d28.36 ±0.14 d
Sun-drying 4.14 ±0.00 f2.94 ±0.00 f15.86 ±0.10 f
Shade-drying 3.72 ±0.00 e2.80 ±0.01 e25.16 ±0.13 e
Microwave-drying 3.11 ±0.00 a2.32 ±0.00 c35.32 ±0.15 a
Oven-drying 3.37 ±0.00 b2.01 ±0.00 a33.47 ±0.10 c
Freeze-drying 3.63 ±0.00 d2.13 ±0.01 b34.74 ±0.14 b
D. sublaevigata
Fresh 43.78 ±0.00 d2.57 ±0.00 c14.06 ±0.10 e
Sun-drying 4.33 ±0.00 f2.76 ±0.00 d6.40 ±0.12 f
Shade-drying 4.12 ±0.00 e2.58 ±0.01 c15.50 ±0.01 d
Microwave-drying 3.36 ±0.00 c2.38 ±0.01 b19.50 ±0.12 c
Oven-drying 3.16 ±0.00 b2.22 ±0.00 a24.60 ±0.06 b
Freeze-drying 3.05 ±0.00 a2.38 ±0.01 b31.23 ±0.11 a
G. levis
Fresh 44.42 ±0.01 f3.09 ±0.00 d29.35 ±0.14 c
Sun-drying 4.36 ±0.00 e3.47 ±0.00 f18.26 ±0.17 e
Shade-drying 4.11 ±0.01 d3.22 ±0.01 e22.80 ±0.09 d
Microwave-drying 4.05 ±0.00 c3.05 ±0.01 c34.22 ±0.11 b
Oven-drying 3.37 ±0.00 b2.47 ±0.00 b34.85 ±0.09 a
Freeze-drying 3.23 ±0.00 a2.44 ±0.00 a35.02 ±0.07 a
S. brachycladum
Fresh 43.49 ±0.01 c2.41 ±0.00 c27.40 ±0.07 d
Sun-drying 4.73 ±0.02 f3.10 ±0.01 e13.33 ±0.03 e
Shade-drying 4.45 ±0.01 e3.33 ±0.01 f13.18 ±0.02 f
Microwave-drying 3.71 ±0.01 d2.36 ±0.00 b30.22 ±0.08 b
Oven-drying 3.28 ±0.01 b2.12 ±0.01 a28.03 ±0.06 c
Freeze-drying 3.23 ±0.00 a2.51 ±0.00 d35.81 ±0.09 a
Trolox 54.09 ±0.00 4.55 ±0.02 –
The values represent the means
±
standard deviations of three replicates. Different letters (within a column)
indicate significant differences (one-way ANOVA, Duncan’s multiple comparison test, p< 0.05).
1
DPPH is
expressed as IC
50
(
µ
g/mL).
2
ABTS is expressed as IC
50
(
µ
g/mL).
3
FRAP is expressed as mg Trolox equivalent
to 1 g of dried sample (mg TE/g).
4
Fresh sample is expressed as a control variable.
5
Trolox is expressed as
a positive control.
Freeze-dried aqueous leaf extracts of G. levis,G. scortechinii, and S. zollingeri exhibited
a higher DPPH yield than ethanolic extracts [
30
]. Fargesia robusta (clumping bamboo) also
Molecules 2022,27, 6458 6 of 23
demonstrated the highest antioxidant capacity for DPPH when its aqueous methanolic
leaf extract was freeze-dried [
31
]. In another study, Kozlowska et al. [
32
] noted that the
antiradical properties of freeze-dried herbal materials (coriander, tarragon, lovage, and
Indian borage) possessed considerably (p< 0.05) superior DPPH scavenging abilities than
the fresh raw material. Nonetheless, the microwave-dried B. vulgaris sample in this study
was subjected to higher microwave power or temperature, thus resulting in increased
antioxidant activity and TPC [33,34].
2.2.2. The 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) Assay
In addition to DPPH, 2,2
0
-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is
one of the most commonly employed assessments to evaluate the antioxidant activities of
plant extracts, foods, and unique compounds [
35
]. The obtained IC
50
value reflects the an-
tioxidant activities of test samples because it records the concentration required to produce
50% inhibition. Accordingly, the lower the IC50 value, the higher the antioxidant activity.
Table 2lists the ABTS values of the bamboo samples evaluated in the current study.
The samples dried under the sun yielded the lowest antioxidant activity, excluding the
fresh sample. The IC
50
values of the B. multiplex,B. tuldoides,B. vulgaris,D. sublaevigata,
and G. levis samples were the most significant, whereas the shade-dried S. brachycladum
contributed the highest IC
50
value. Saifullah et al. [
36
] reported that the antioxidant capacity
measured via ABTS of the sun- and shade-dried lemon myrtle exhibited the lowest yield
compared with hot-air-, vacuum-, and freeze-dried specimens.
The highest antioxidant activities or the lowest IC
50
values were recorded by the oven-
dried B. multiplex,B. tuldoides,B. vulgaris,D. sublaevigata, and S. brachycladum. Nevertheless,
only the G. levis sample exhibited the least IC
50
value when freeze-dried. Chuyen et al. [
37
]
documented that the peels of Gac fruits which were hot-air-dried at 60 and 80
◦
C and
vacuum-dried at 50
◦
C exhibited the highest ABTS antioxidant capacities. Consequently,
hot-air- and vacuum-drying at 80 and 50
◦
C were recommended for drying Gac peel.
Hot-air-, vacuum-, and freeze-drying methods were recommended to preserve the ABTS
antioxidant activity of lemon myrtle because these techniques produced significant values
compared with other methods [
36
]. Hot-air-drying at 40–60
◦
C was recommended for
herbs [
38
], which explained the highest antioxidant activity in ABTS of the oven-dried
samples in this study that was conducted at 50 ◦C.
2.2.3. The Ferric Reducing Antioxidant Power Assay
The reducing properties of the samples in this study were assessed via the ferric
reducing antioxidant power (FRAP) assay. The results varied significantly (p< 0.05) be-
tween treatments applied (see Table 2). The sun-dried B. multiplex,B. tuldoides,B. vulgaris,
D. sublaevigata
, and G. levis samples, and shade-dried S. brachycladum samples, exhibited
considerably reduced FRAP contents. The results aligned with one report which demon-
strated that traditionally dried spearmints, particularly sun- and shade-dried extracts,
demonstrated a notably diminished FRAP compared with freeze-dried samples [
39
]. Tradi-
tional drying methods, such as sun- and shade-drying, possess numerous drawbacks due
to their inability to produce the high-quality standards required for medicinal plants [24].
Freeze-dried aqueous extracts of B. multiplex,D. sublaevigata,G. levis, and S. brachycladum
resulted in the highest FRAP amount. Kong et al. [
40
] noted that the reducing power of the
Clinacanthus nutans leaf extract when freeze-dried rose with increased value (mg TE/g),
where the highest level was noted in the aqueous extract (10.07
±
0.10), followed by the
ethanolic (8.34
±
0.14) and acetone (3.24
±
0.30) samples. Nevertheless, the microwave-
dried B. tuldoides and B. vulgaris specimens in the current study recorded the highest FRAP
yield. Similarly, Lasano et al. [
41
] recommended microwaving fermented and unfermented
Strobilanthes crispus tea to obtain preferable antioxidant capacities, including FRAP and
DPPH. The antioxidant index proposed by the report might also be employed as a new
marker in determining the optimal drying method for varying food products [41].
Molecules 2022,27, 6458 7 of 23
2.3. The Correlation between Phytochemical Contents and Antioxidant Capacities
The antioxidant attributes of a plant extract are often associated with its polyphenols
content; therefore, the correlation between its phytochemical contents and the antioxidant
capacity of the selected bamboo extracts in this study dried with different methods was
analysed, and the outcomes are summarised in Table 3. The negative DPPH and ABTS
values documented by all samples might also correspond to the IC
50
value, because it is
inversely proportional to the free radical scavenging activity of the samples, indicating that
low IC
50
samples possessed high antioxidant activity. Overall, the correlation coefficient
values of the specimens demonstrated significant (p< 0.01) and moderate correlations
between TPC-DPPH, TFC-DPPH, TPC-ABTS, TFC-ABTS, and TFC-FRAP.
Table 3.
The Pearson correlation between the phytochemical contents and antioxidant capacities of
the dried bamboo extracts.
Phytochemical
Antioxidant Capacity
DPPH ABTS FRAP
Rp-Value R p-Value R p-Value
TPC −0.40 ** 0.00 −0.42 ** 0.00 −0.05 0.64
TFC −0.45 ** 0.00 −0.39 ** 0.00 0.42 ** 0.00
** Correlation is significant at the 0.01 level (2-tailed).
The DPPH, ABTS, and FRAP R-values of the Pearson correlation coefficient for the TFC
were
−
0.45,
−
0.39, and 0.42, respectively, revealing that flavonoids were the primary contrib-
utor to the antioxidant capacities of DPPH, ABTS, and FRAP. Similar results were observed
by Ni et al. [
21
], with a moderate correlation between TFC and DPPH (R = 0.45) and a strong
association between TFC and FRAP (R = 0.81) in
Pleioblastus kongosanensis f. aureostriatus
and
Shibataea chinensis. Moreover, the TPC in this study documented a moderate association
with antioxidant activities in DPPH and ABTS, with R-values of
−
0.40 and
−
0.42, respec-
tively. Correspondingly, Hu et al. [
42
] reported a strong relationship between TPC and
DPPH (R = 0.74) in Phyllostachys spp. leaves. Pande et al. [
43
] described similar findings for
B. nutans leaf extracts under different extraction conditions.
The TPC-FRAP revealed no association with the Pearson correlation coefficient, which
recorded an R-value of
−
0.05. The results indicated that TPC did not contribute to the
high antioxidant capacity of FRAP. The findings were similar to the report by Ni et al. [
21
],
who demonstrated a weak correlation between TPC and FRAP (R = 0.12). By comparing
the correlation coefficient of the R-values, it is possible to suggest that the phenolic and
flavonoid groups were slightly responsible for the antioxidant activities of DPPH, ABTS,
and FRAP, as stated by Ouyang et al. [
44
]. In addition, the weaker correlation may be due
to the fact that phenolics comprise a sizable collection of chemicals with various structures
and antioxidant properties [
45
]. Due to the presence of non-participating elements such
as sugars, flavonoids commonly form bonds with sugar moieties to create glycosides,
which have a lower DPPH scavenging activity than their aglycones or phenolic acids
on
a weight
basis [
45
]. Accordingly, quantifying the contributions of the phenolic and
flavonoid compounds to the total antioxidant activity is necessary to understand the
correlation between them and their connection to the antioxidant activity.
2.4. The BSLA
Statistical analysis of the B. multiplex (freeze-drying), B. tuldoides (freeze-drying),
B. vulgaris
(microwave-drying), D. sublaevigata (freeze-drying), G. levis (freeze-drying), and
S. brachycladum (freeze-drying) samples recorded significant TPC, TFC, DPPH, ABTS, and
FRAP values. The bamboo extracts were further studied for their toxicity tests via the BSLA.
The LC
50
(lethal concentration 50) values of the extracts and the positive control, potassium
dichromate (K2Cr2O7), are presented in Table 4.
Molecules 2022,27, 6458 8 of 23
Table 4.
The mortality percentage and lethality concentration of shrimp nauplii after treatment with
the bamboo extracts.
Samples Concentration (µg/mL) % Mortality LC50 (µg/mL)
K2Cr2O71
1000 100
11.23
100 33
10 33
1 27
B. multiplex
1000 17
3744.85
100 10
10 7
1 0
B. tuldoides
1000 20
2974.47
100 10
10 7
1 0
B. vulgaris
1000 17
3166.15
100 13
10 7
1 0
D. sublaevigata
1000 13
5668.14
100 10
10 7
1 0
G. levis
1000 27
1236.53
100 20
10 10
1 0
S. brachycladum
1000 20
2045.03
100 17
10 10
1 0
1K2Cr2O7was expressed as a positive control.
In the current study, the mortality rate of brine shrimp was proportional to the concen-
tration of test samples evaluated. The B. multiplex,B. tuldoides,B. vulgaris,
D. sublaevigata,
G. levis
, and S. brachycladum extracts exhibited no significant toxicity towards brine shrimps
at LC
50
values of 3744.85, 2974.47, 3166.15, 5668.14, 1236.53, and 2045.03
µ
g/mL, re-
spectively. The positive control, K
2
Cr
2
O
7
, recorded an LC
50
of 11.23
µ
g/mL, indicating
high toxicity.
The BSLA was conducted to determine the functional properties of the selected bam-
boo extracts. Nevertheless, reports on the impacts of drying on the toxicity of aqueous
bamboo extracts worldwide are limited. Consequently, the present study employed the
BSLA as a reliable method for preliminary toxicity assessment of the extracts. Moreover, this
study compared the BSLA results of the aqueous bamboo extracts with aqueous medicinal
plant leaves extracts.
One investigation observed that among the evaluated shade-dried extracts of
Pentapetes phoenicea, the chloroform and ethyl acetate extracts were weakly toxic with
LC
50
values of 659.8 and 928.9
µ
g/mL, respectively [
46
]. Conversely, the hexane and
aqueous extracts were non-toxic, recording LC
50
values of 1293.6 and 1929.2
µ
g/mL,
respectively [
46
]. Shawa et al. [
47
] also reported that an aqueous Senna singuena leaves
extract air-dried at room temperature did not demonstrate significant toxicity after 24 h.
Furthermore, the BSLA values of all Phragmanthera capitata leaf solvent extracts (including
aqueous extract) were not toxic at LC50 > 1000 µg/mL [48].
In conclusion, bamboo extracts could be considered safe for consumption as herbal
medicine and could potentially be developed as herbal tea. Nonetheless, the non-toxic
Molecules 2022,27, 6458 9 of 23
attributes exhibited by the plant could be discouraging in treating and managing cancer or
tumour alternatives, because BSLA is commonly employed as an indicator for preliminary
bioactivity screening, including for anticancer [49].
2.5. Chemical Constituents
Traditional medicine uses well-known natural products in the form of secondary
metabolites derived from a wide range of natural sources. These specialised metabolites
found in fungi, plants, and marine creatures function as a formidable armoury against
biotic and abiotic stressors. In addition, medicinal chemists utilise natural products as
structural scaffolds to synthesise new medications with enhanced pharmacological efficacy
and safety [
50
–
52
]. Nevertheless, metabolite discovery remains a significant bottleneck
in traditional medicine [
53
]. As a result of multiple erroneous identifications of small
compounds, bioactivity investigations of traditional medicines have adopted an evidence-
based approach [
53
]. Thus, LC-MS/MS was used to further quantify B. multiplex (freeze-
drying), B. tuldoides (freeze-drying), B. vulgaris (microwave-drying), D. sublaevigata (freeze-
drying), G. levis (freeze-drying), and S. brachycladum (freeze-drying) for the purpose of
profiling their bioactive compounds that contribute to antioxidant properties and functional
pharmaceutical applications.
The liquid chromatogram of the LC-MS/MS analysis for B. multiplex is shown in
Figure 1. The compounds identified in the B. multiplex are tabulated in Table 5along with
their molecular formula, molecular weight, and m/zvalue. In the present study, 18 detected
peaks showed significance. The highest peak was found to be felodipine (peak 53). In
B. multiplex,
alkaloid compounds identified as caffeine were found in peaks 28, 30, 32, and
37. Other compounds found in B. multiplex were: L-histidine (peak 9); pararosaniline (peak
39); felodipine (peak 45); and phytosphingosine (peak 60).
Molecules 2022, 27, x FOR PEER REVIEW 9 of 24
1
K
2
Cr
2
O
7
was expressed as a positive control.
The BSLA was conducted to determine the functional properties of the selected bam-
boo extracts. Nevertheless, reports on the impacts of drying on the toxicity of aqueous
bamboo extracts worldwide are limited. Consequently, the present study employed the
BSLA as a reliable method for preliminary toxicity assessment of the extracts. Moreover,
this study compared the BSLA results of the aqueous bamboo extracts with aqueous me-
dicinal plant leaves extracts.
One investigation observed that among the evaluated shade-dried extracts of Pen-
tapetes phoenicea, the chloroform and ethyl acetate extracts were weakly toxic with LC
50
values of 659.8 and 928.9 µg/mL, respectively [46]. Conversely, the hexane and aqueous
extracts were non-toxic, recording LC
50
values of 1293.6 and 1929.2 µg/mL, respectively
[46]. Shawa et al. [47] also reported that an aqueous Senna singuena leaves extract air-dried
at room temperature did not demonstrate significant toxicity after 24 h. Furthermore, the
BSLA values of all Phragmanthera capitata leaf solvent extracts (including aqueous extract)
were not toxic at LC
50
> 1000 µg/mL [48].
In conclusion, bamboo extracts could be considered safe for consumption as herbal
medicine and could potentially be developed as herbal tea. Nonetheless, the non-toxic
attributes exhibited by the plant could be discouraging in treating and managing cancer
or tumour alternatives, because BSLA is commonly employed as an indicator for prelim-
inary bioactivity screening, including for anticancer [49].
2.5. Chemical Constituents
Traditional medicine uses well-known natural products in the form of secondary me-
tabolites derived from a wide range of natural sources. These specialised metabolites
found in fungi, plants, and marine creatures function as a formidable armoury against
biotic and abiotic stressors. In addition, medicinal chemists utilise natural products as
structural scaffolds to synthesise new medications with enhanced pharmacological effi-
cacy and safety [50–52]. Nevertheless, metabolite discovery remains a significant bottle-
neck in traditional medicine [53]. As a result of multiple erroneous identifications of small
compounds, bioactivity investigations of traditional medicines have adopted an evidence-
based approach [53]. Thus, LC-MS/MS was used to further quantify B. multiplex (freeze-
drying), B. tuldoides (freeze-drying), B. vulgaris (microwave-drying), D. sublaevigata
(freeze-drying), G. levis (freeze-drying), and S. brachycladum (freeze-drying) for the pur-
pose of profiling their bioactive compounds that contribute to antioxidant properties and
functional pharmaceutical applications.
The liquid chromatogram of the LC-MS/MS analysis for B. multiplex is shown in Fig-
ure 1. The compounds identified in the B. multiplex are tabulated in Table 5 along with
their molecular formula, molecular weight, and m/z value. In the present study, 18 de-
tected peaks showed significance. The highest peak was found to be felodipine (peak 53).
In B. multiplex, alkaloid compounds identified as caffeine were found in peaks 28, 30, 32,
and 37. Other compounds found in B. multiplex were: L-histidine (peak 9); pararosaniline
(peak 39); felodipine (peak 45); and phytosphingosine (peak 60).
Figure 1. LC-MS/MS chromatogram of B. multiplex.
Figure 1. LC-MS/MS chromatogram of B. multiplex.
The liquid chromatogram of the LC-MS/MS analysis for B. tuldoides is shown in
Figure 2. The compounds identified in B. tuldoides are tabulated in Table 6along with their
molecular formula, molecular weight, and m/zvalue. In the present study, 14 detected
peaks showed significance. The highest peak was found to be felodipine (peak 35). In
B. tuldoides
, alkaloid compounds identified as sparteine and papaverine were found in
peaks 5 and 16, respectively. Other compounds found in B. tuldoides were: PET-cGMP
(peak 12)
; naloxone (peak 14); thiopental (peak 22); cyproheptadine (peak 24); loprazolam
(peak 26)
; difenoconazole (peak 28); RP-8-pCPT-cGMPS (peak 31); and felodipine
(peak 44)
.
The liquid chromatogram of the LC-MS/MS analysis for B. vulgaris is shown in
Figure 3
. The compounds identified in B. vulgaris are tabulated in Table 7along with their
molecular formula, molecular weight, and m/zvalue. In the present study,
13 detected
peaks showed significance. The highest peak was found to be felodipine (peak 42). In
B. vulgaris
, alkaloid compounds identified as papaverine were found in peak 11. Other com-
pounds found in B. vulgaris were: econazole (peak 3); pimozide (peak 9); cyproheptadine
(peak 15); bisacodyl (peak 19); loprazolam (peak 25); difenoconazole (peak 36); felodipine
(peak 51); and cinchocaine (peak 58).
Molecules 2022,27, 6458 10 of 23
Table 5. The compounds identified in B. multiplex.
Peak RT (min) Identified
Compounds
Molecular
Formula
Molecular
Weight m/z
3 0.5 Unidentified – 199.9663 200.9736
9 0.6 L-Histidine C6H9N3O2155.0354 156.0427
25 2.2 Unidentified – 445.2898 446.2971
28 4.8 Caffeine C8H10N4O2577.3688 578.3760
29 7.4 Unidentified – 452.3372 453.3445
30 13.9 Caffeine C8H10N4O2550.1329 551.1402
31 18.2 Unidentified – 534.1379 535.1452
32 23.0 Caffeine C8H10N4O2534.1378 535.1451
33 26.9 Unidentified – 700.4868 701.4941
37 29.2 Caffeine C8H10N4O2428.1840 429.1913
39 38.2 Pararosaniline C19 H17N3287.2837 288.2909
41 39.7 Unidentified – 315.2784 316.2857
43 42.2 Unidentified – 315.3145 316.3218
44 43.7 Unidentified – 315.3144 316.3217
45 77.2 Felodipine C18H19 Cl2NO4337.3346 338.3419
53 78.0 Felodipine C18H19 Cl2NO4337.3365 338.3438
60 79.5 Phytosphingosine C18H39NO3337.3346 338.3419
64 82.1 Unidentified – 343.2721 344.2794
Molecules 2022, 27, x FOR PEER REVIEW 10 of 24
Table 5. The compounds identified in B. multiplex.
Peak RT (min) Identified Compounds Molecular Formula Molecular Weight m/z
3 0.5 Unidentified – 199.9663
200.9736
9 0.6
L-Histidine C
6
H
9
N
3
O
2
155.0354
156.0427
25 2.2 Unidentified – 445.2898 446.2971
28 4.8 Caffeine C
8
H
10
N
4
O
2
577.3688 578.3760
29 7.4 Unidentified – 452.3372 453.3445
30 13.9 Caffeine C
8
H
10
N
4
O
2
550.1329 551.1402
31 18.2 Unidentified – 534.1379 535.1452
32 23.0 Caffeine C
8
H
10
N
4
O
2
534.1378 535.1451
33 26.9 Unidentified – 700.4868 701.4941
37 29.2 Caffeine C
8
H
10
N
4
O
2
428.1840 429.1913
39 38.2 Pararosaniline C
19
H
17
N
3
287.2837 288.2909
41 39.7 Unidentified – 315.2784 316.2857
43 42.2 Unidentified – 315.3145 316.3218
44 43.7 Unidentified – 315.3144 316.3217
45 77.2 Felodipine C
18
H
19
Cl
2
NO
4
337.3346 338.3419
53 78.0 Felodipine C
18
H
19
Cl
2
NO
4
337.3365
338.3438
60 79.5 Phytosphingosine C
18
H
39
NO
3
337.3346 338.3419
64 82.1 Unidentified – 343.2721 344.2794
The liquid chromatogram of the LC-MS/MS analysis for B. tuldoides is shown in Fig-
ure 2. The compounds identified in B. tuldoides are tabulated in Table 6 along with their
molecular formula, molecular weight, and m/z value. In the present study, 14 detected
peaks showed significance. The highest peak was found to be felodipine (peak 35). In B.
tuldoides, alkaloid compounds identified as sparteine and papaverine were found in peaks
5 and 16, respectively. Other compounds found in B. tuldoides were: PET-cGMP (peak 12);
naloxone (peak 14); thiopental (peak 22); cyproheptadine (peak 24); loprazolam (peak 26);
difenoconazole (peak 28); RP-8-pCPT-cGMPS (peak 31); and felodipine (peak 44).
Figure 2. LC-MS/MS chromatogram of B. tuldoides.
Table 6. The compounds identified in B. tuldoides.
Peak RT (min) Identified Compounds Molecular Formula Molecular Weight m/z
5 0.6 Sparteine C
15
H
26
N
2
234.1583
235.1656
12 2.5
PET-cGMP C
18
H
15
N
5
O
7
PNa 113.0840
114.0913
14 5.7 Naloxone C
19
H
21
NO
4
327.2521 328.2594
15 7.6 Unidentified – 452.3361 453.3434
16 25.0 Papaverine C
20
H
21
NO
4
678.5037 340.2591
22 27.7 Thiopental C
11
H
18
N
2
O
2
S 791.5873 396.8009
24 38.0 Cyproheptadine C
21
H
21
N 287.2827 288.2900
26 42.0 Loprazolam C
23
H
21
ClN
6
O
3
315.3139 316.3211
28 77.5 Difenoconazole C
19
H
17
Cl
2
N
3
O
3
309.3035 310.3108
31 78.0 RP-8-pCPT-cGMPS C
16
H
14
ClN
5
O
6
PS
2
Na 311.3193 312.3265
35 78.0 Felodipine C
18
H
19
Cl
2
NO
4
337.3367 338.3439
44 79.5 Felodipine C
18
H
19
Cl
2
NO
4
337.3348 338.3421
49 81.2 Unidentified – 337.3347 338.3419
Figure 2. LC-MS/MS chromatogram of B. tuldoides.
Table 6. The compounds identified in B. tuldoides.
Peak RT (min) Identified
Compounds Molecular Formula Molecular
Weight m/z
5 0.6 Sparteine C15 H26N2234.1583 235.1656
12 2.5 PET-cGMP C18 H15N5O7PNa 113.0840 114.0913
14 5.7 Naloxone C19H21NO4327.2521 328.2594
15 7.6 Unidentified – 452.3361 453.3434
16 25.0 Papaverine C20H21NO4678.5037 340.2591
22 27.7 Thiopental C11H18 N2O2S 791.5873 396.8009
24 38.0 Cyproheptadine C21 H21N 287.2827 288.2900
26 42.0 Loprazolam C23H21ClN6O3315.3139 316.3211
28 77.5 Difenoconazole C19 H17Cl2N3O3309.3035 310.3108
31 78.0 RP-8-pCPT-cGMPS C16H14ClN5O6PS2Na 311.3193 312.3265
35 78.0 Felodipine C18H19 Cl2NO4337.3367 338.3439
44 79.5 Felodipine C18H19 Cl2NO4337.3348 338.3421
49 81.2 Unidentified – 337.3347 338.3419
52 82.8 Unidentified – 225.9441 226.9514
Molecules 2022,27, 6458 11 of 23
Molecules 2022, 27, x FOR PEER REVIEW 11 of 24
52 82.8 Unidentified – 225.9441
226.9514
The liquid chromatogram of the LC-MS/MS analysis for B. vulgaris is shown in Figure
3. The compounds identified in B. vulgaris are tabulated in Table 7 along with their mo-
lecular formula, molecular weight, and m/z value. In the present study, 13 detected peaks
showed significance. The highest peak was found to be felodipine (peak 42). In B. vulgaris,
alkaloid compounds identified as papaverine were found in peak 11. Other compounds
found in B. vulgaris were: econazole (peak 3); pimozide (peak 9); cyproheptadine (peak
15); bisacodyl (peak 19); loprazolam (peak 25); difenoconazole (peak 36); felodipine (peak
51); and cinchocaine (peak 58).
Figure 3. LC-MS/MS chromatogram of B. vulgaris.
Table 7. The compounds identified in B. vulgaris.
Peak RT (min) Identified Compounds Molecular Formula Molecular Weight m/z
3 0.5 Econazole C
18
H
15
Cl
3
N
2
O 155.0349
156.0422
7 0.7
Unidentified – 200.0322
201.0394
9 3.1 Pimozide C
28
H
29
F
2
N
3
O 489.3156 490.3229
10 7.6 Unidentified – 452.3377 453.3449
11 26.9 Papaverine C
20
H
21
NO
4
678.5046 340.2596
15 37.9 Cyproheptadine C
21
H
21
N 287.2832 288.2905
19 39.6 Bisacodyl C
22
H
19
NO
4
361.1714 362.1787
25 41.9 Loprazolam C
23
H
21
ClN
6
O
3
315.3143 316.3216
36 77.5 Difenoconazole C
19
H
17
Cl
2
N
3
O
3
337.3351 338.3423
42 78.0 Felodipine C
18
H
19
Cl
2
NO
4
320.3085 321.3158
51 79.5 Felodipine C
18
H
19
Cl
2
NO
4
337.3352 338.3425
52 79.8 Unidentified – 343.2730 344.2802
58 81.4 Cinchocaine C
20
H
29
N
3
O
2
337.3349
338.3422
The liquid chromatogram of the LC-MS/MS analysis for D. sublaevigata is shown in
Figure 4. The compounds identified in D. sublaevigata are tabulated in Table 8 along with
their molecular formula, molecular weight, and m/z value. In the present study, 15 de-
tected peaks showed significance. The highest peak was found to be RP-8-pCPT-cGMPS
(peak 40). In D. sublaevigata, alkaloid compounds identified as papaverine were found in
peaks 22 and 24. Other compounds found in D. sublaevigata were: phenytoin (peak 5);
perazine (peak 12); penconazole (peak 19); cyproheptadine (peak 29); RP-8-pCPT-cGMPS
(peak 36); felodipine (peak 49); and cinchocaine (peaks 51 and 56).
Figure 3. LC-MS/MS chromatogram of B. vulgaris.
Table 7. The compounds identified in B. vulgaris.
Peak RT (min) Identified
Compounds
Molecular
Formula
Molecular
Weight m/z
3 0.5 Econazole C18 H15Cl3N2O 155.0349 156.0422
7 0.7 Unidentified – 200.0322 201.0394
9 3.1 Pimozide C28H29F2N3O 489.3156 490.3229
10 7.6 Unidentified – 452.3377 453.3449
11 26.9 Papaverine C20H21NO4678.5046 340.2596
15 37.9 Cyproheptadine C21H21N 287.2832 288.2905
19 39.6 Bisacodyl C22H19NO4361.1714 362.1787
25 41.9 Loprazolam C23 H21ClN6O3315.3143 316.3216
36 77.5 Difenoconazole C19H17Cl2N3O3337.3351 338.3423
42 78.0 Felodipine C18H19 Cl2NO4320.3085 321.3158
51 79.5 Felodipine C18H19 Cl2NO4337.3352 338.3425
52 79.8 Unidentified – 343.2730 344.2802
58 81.4 Cinchocaine C20H29N3O2337.3349 338.3422
The liquid chromatogram of the LC-MS/MS analysis for D. sublaevigata is shown in
Figure 4. The compounds identified in D. sublaevigata are tabulated in Table 8along with
their molecular formula, molecular weight, and m/zvalue. In the present study, 15 detected
peaks showed significance. The highest peak was found to be RP-8-pCPT-cGMPS (
peak 40
).
In D. sublaevigata, alkaloid compounds identified as papaverine were found in peaks 22 and
24. Other compounds found in D. sublaevigata were: phenytoin (peak 5); perazine (peak 12);
penconazole (peak 19); cyproheptadine (peak 29); RP-8-pCPT-cGMPS (peak 36); felodipine
(peak 49); and cinchocaine (peaks 51 and 56).
Molecules 2022, 27, x FOR PEER REVIEW 11 of 24
52 82.8 Unidentified – 225.9441
226.9514
The liquid chromatogram of the LC-MS/MS analysis for B. vulgaris is shown in Figure
3. The compounds identified in B. vulgaris are tabulated in Table 7 along with their mo-
lecular formula, molecular weight, and m/z value. In the present study, 13 detected peaks
showed significance. The highest peak was found to be felodipine (peak 42). In B. vulgaris,
alkaloid compounds identified as papaverine were found in peak 11. Other compounds
found in B. vulgaris were: econazole (peak 3); pimozide (peak 9); cyproheptadine (peak
15); bisacodyl (peak 19); loprazolam (peak 25); difenoconazole (peak 36); felodipine (peak
51); and cinchocaine (peak 58).
Figure 3. LC-MS/MS chromatogram of B. vulgaris.
Table 7. The compounds identified in B. vulgaris.
Peak RT (min) Identified Compounds Molecular Formula Molecular Weight m/z
3 0.5 Econazole C
18
H
15
Cl
3
N
2
O 155.0349
156.0422
7 0.7
Unidentified – 200.0322
201.0394
9 3.1 Pimozide C
28
H
29
F
2
N
3
O 489.3156 490.3229
10 7.6 Unidentified – 452.3377 453.3449
11 26.9 Papaverine C
20
H
21
NO
4
678.5046 340.2596
15 37.9 Cyproheptadine C
21
H
21
N 287.2832 288.2905
19 39.6 Bisacodyl C
22
H
19
NO
4
361.1714 362.1787
25 41.9 Loprazolam C
23
H
21
ClN
6
O
3
315.3143 316.3216
36 77.5 Difenoconazole C
19
H
17
Cl
2
N
3
O
3
337.3351 338.3423
42 78.0 Felodipine C
18
H
19
Cl
2
NO
4
320.3085 321.3158
51 79.5 Felodipine C
18
H
19
Cl
2
NO
4
337.3352 338.3425
52 79.8 Unidentified – 343.2730 344.2802
58 81.4 Cinchocaine C
20
H
29
N
3
O
2
337.3349
338.3422
The liquid chromatogram of the LC-MS/MS analysis for D. sublaevigata is shown in
Figure 4. The compounds identified in D. sublaevigata are tabulated in Table 8 along with
their molecular formula, molecular weight, and m/z value. In the present study, 15 de-
tected peaks showed significance. The highest peak was found to be RP-8-pCPT-cGMPS
(peak 40). In D. sublaevigata, alkaloid compounds identified as papaverine were found in
peaks 22 and 24. Other compounds found in D. sublaevigata were: phenytoin (peak 5);
perazine (peak 12); penconazole (peak 19); cyproheptadine (peak 29); RP-8-pCPT-cGMPS
(peak 36); felodipine (peak 49); and cinchocaine (peaks 51 and 56).
Figure 4. LC-MS/MS chromatogram of D. sublaevigata.
Molecules 2022,27, 6458 12 of 23
Table 8. The compounds identified in D. sublaevigata.
Peak RT (min) Identified
Compounds Molecular Formula Molecular
Weight m/z
5 0.5 Phenytoin C15 H12N2O2155.0349 156.0421
12 2.4 Perazine C20H25N3S 339.2523 340.2596
15 5.3 Unidentified – 474.3177 475.3250
18 7.7 Unidentified – 452.3365 453.3438
19 13.2 Penconazole C13H15 Cl2N3565.4204 566.4276
22 25.0 Papaverine C20 H21NO4678.5038 679.5111
24 27.0 Papaverine C20 H21NO4678.5026 340.2586
25 27.7 Unidentified – 813.5703 814.5776
29 38.0 Cyproheptadine C21 H21N 287.2825 288.2897
31 42.0 Unidentified – 315.3136 316.3209
36 78.0 RP-8-pCPT-cGMPS C16H14 ClN5O6PS2Na 311.3185 312.3258
40 78.0 RP-8-pCPT-cGMPS C16H14 ClN5O6PS2Na 320.3077 321.3149
49 79.5 Felodipine C18H19 Cl2NO4337.3339 338.3412
51 80.0 Cinchocaine C20 H29N3O2343.2714 344.2787
56 82.1 Cinchocaine C20 H29N3O2343.2717 344.2789
The liquid chromatogram of the LC-MS/MS analysis for G. levis is shown in Figure 5.
The compounds identified in G. levis are tabulated in Table 9along with their molecular
formula, molecular weight, and m/zvalue. In the present study, 13 detected peaks showed
significance. The highest peak was found to be RP-8-pCPT-cGMPS (peak 32). In G. levis,
alkaloid compounds identified as papaverine were found in peak 15. Other compounds
found in G. levis were: L-histidine (peak 3); PET-cGMP (peak 10); naloxone (peak 12);
cyproheptadine (peak 22); loprazolam (peak 24); RP-8-pCPT-cGMPS (peak 29); felodipine
(peak 42); cinchocaine (peak 43); and amphetamine (peak 51).
Molecules 2022, 27, x FOR PEER REVIEW 12 of 24
Figure 4. LC-MS/MS chromatogram of D. sublaevigata.
Table 8. The compounds identified in D. sublaevigata.
Peak RT (min) Identified Compounds Molecular Formula Molecular Weight m/z
5 0.5 Phenytoin C
15
H
12
N
2
O
2
155.0349
156.0421
12 2.4
Perazine C
20
H
25
N
3
S 339.2523
340.2596
15 5.3 Unidentified – 474.3177 475.3250
18 7.7 Unidentified – 452.3365 453.3438
19 13.2 Penconazole C
13
H
15
Cl
2
N
3
565.4204 566.4276
22 25.0 Papaverine C
20
H
21
NO
4
678.5038 679.5111
24 27.0 Papaverine C
20
H
21
NO
4
678.5026 340.2586
25 27.7 Unidentified – 813.5703 814.5776
29 38.0 Cyproheptadine C
21
H
21
N 287.2825 288.2897
31 42.0 Unidentified – 315.3136 316.3209
36 78.0 RP-8-pCPT-cGMPS C
16
H
14
ClN
5
O
6
PS
2
Na 311.3185 312.3258
40 78.0 RP-8-pCPT-cGMPS C
16
H
14
ClN
5
O
6
PS
2
Na 320.3077 321.3149
49 79.5 Felodipine C
18
H
19
Cl
2
NO
4
337.3339 338.3412
51 80.0 Cinchocaine C
20
H
29
N
3
O
2
343.2714 344.2787
56 82.1 Cinchocaine C
20
H
29
N
3
O
2
343.2717
344.2789
The liquid chromatogram of the LC-MS/MS analysis for G. levis is shown in Figure 5.
The compounds identified in G. levis are tabulated in Table 9 along with their molecular
formula, molecular weight, and m/z value. In the present study, 13 detected peaks showed
significance. The highest peak was found to be RP-8-pCPT-cGMPS (peak 32). In G. levis,
alkaloid compounds identified as papaverine were found in peak 15. Other compounds
found in G. levis were: L-histidine (peak 3); PET-cGMP (peak 10); naloxone (peak 12); cy-
proheptadine (peak 22); loprazolam (peak 24); RP-8-pCPT-cGMPS (peak 29); felodipine
(peak 42); cinchocaine (peak 43); and amphetamine (peak 51).
Figure 5. LC-MS/MS chromatogram of G. levis.
Table 9. The compounds identified in G. levis.
Peak RT (min) Identified Compounds Molecular Formula Molecular Weight m/z
3 0.5 L-Histidine C
6
H
9
N
3
O
2
155.0347
156.0420
10 2.5
PET-cGMP C
18
H
15
N
5
O
7
PNa 113.0840
114.0913
12 5.7 Naloxone C
19
H
21
NO
4
327.2530 328.2602
13 7.6 Unidentified – 452.3369 453.3442
15 25.0 Papaverine C
20
H
21
NO
4
678.5056 340.2601
18 27.7 Unidentified – 813.5731 814.5803
22 38.0 Cyproheptadine C
21
H
21
N 287.2833 288.2906
24 42.0 Loprazolam C
23
H
21
ClN
6
O
3
315.3148 316.3221
29 78.0 RP-8-pCPT-cGMPS C
16
H
14
ClN
5
O
6
PS
2
Na 311.3195 312.3268
32 78.0 RP-8-pCPT-cGMPS C
16
H
14
ClN
5
O
6
PS
2
Na 674.6705 675.6778
42 79.5 Felodipine C
18
H
19
Cl
2
NO
4
337.3351 338.3424
43 80.0 Cinchocaine C
20
H
29
N
3
O
2
343.2728 344.2801
51 82.8 Amphetamine C
9
H
13
N 225.9441
226.9513
Figure 5. LC-MS/MS chromatogram of G. levis.
Table 9. The compounds identified in G. levis.
Peak RT (min) Identified
Compounds Molecular Formula Molecular
Weight m/z
3 0.5 L-Histidine C6H9N3O2155.0347 156.0420
10 2.5 PET-cGMP C18 H15N5O7PNa 113.0840 114.0913
12 5.7 Naloxone C19H21NO4327.2530 328.2602
13 7.6 Unidentified – 452.3369 453.3442
15 25.0 Papaverine C20 H21NO4678.5056 340.2601
18 27.7 Unidentified – 813.5731 814.5803
22 38.0 Cyproheptadine C21 H21N 287.2833 288.2906
24 42.0 Loprazolam C23H21ClN6O3315.3148 316.3221
29 78.0 RP-8-pCPT-cGMPS C16H14 ClN5O6PS2Na 311.3195 312.3268
32 78.0 RP-8-pCPT-cGMPS C16H14 ClN5O6PS2Na 674.6705 675.6778
42 79.5 Felodipine C18H19 Cl2NO4337.3351 338.3424
43 80.0 Cinchocaine C20 H29N3O2343.2728 344.2801
51 82.8 Amphetamine C9H13N 225.9441 226.9513
The liquid chromatogram of the LC-MS/MS analysis for S. brachycladum is shown
in Figure 6. The compounds identified in S. brachycladum are tabulated in Table 10 along
with their molecular formula, molecular weight, and m/zvalue. In the present study,
13 detected
peaks showed significance. The highest peak was found to be felodipine
(
peak 35
). However, no alkaloids were found in S. brachycladum. Other compounds found
Molecules 2022,27, 6458 13 of 23
in S. brachycladum were: amphetamine (peak 7); naloxone (peak 10); perazine (peak 15);
cyproheptadine (peak 24); difenoconazole (peak 27); RP-8-pCPT-cGMPS (peak 32); and
felodipine (peak 47).
Molecules 2022, 27, x FOR PEER REVIEW 13 of 24
The liquid chromatogram of the LC-MS/MS analysis for S. brachycladum is shown in
Figure 6. The compounds identified in S. brachycladum are tabulated in Table 10 along with
their molecular formula, molecular weight, and m/z value. In the present study, 13 de-
tected peaks showed significance. The highest peak was found to be felodipine (peak 35).
However, no alkaloids were found in S. brachycladum. Other compounds found in S.
brachycladum were: amphetamine (peak 7); naloxone (peak 10); perazine (peak 15); cypro-
heptadine (peak 24); difenoconazole (peak 27); RP-8-pCPT-cGMPS (peak 32); and felodi-
pine (peak 47).
Figure 6. LC-MS/MS chromatogram of S. brachycladum.
Table 10. The compounds identified in S. brachycladum.
Peak RT (min) Identified Compounds Molecular Formula Molecular Weight m/z
3 0.5 Unidentified – 155.0348
156.0421
7 2.5
Amphetamine C
9
H
13
N 113.0840
114.0913
10 5.7 Naloxone C
19
H
21
NO
4
327.2523 328.2596
11 7.6 Unidentified – 452.3367 453.3440
15 25.0 Perazine C
20
H
25
N
3
S 678.5043 340.2594
18 27.7 Unidentified – 829.5351 415.7748
24 37.4 Cyproheptadine C
21
H
21
N 287.2828 288.2901
25 41.3 Unidentified – 315.3141 316.3214
27 77.5 Difenoconazole C
19
H
17
Cl
2
N
3
O
3
309.3037 310.3110
32 78.0 RP-8-pCPT-cGMPS C
16
H
14
ClN
5
O
6
PS
2
Na 311.3194 312.3267
35 78.0 Felodipine C
18
H
19
Cl
2
NO
4
337.3365 338.3437
47 79.5 Felodipine C
18
H
19
Cl
2
NO
4
337.3351 338.3423
52 81.4 Unidentified – 343.2726
344.2799
In brief, on the basis of the notable differences, the alkaloid compounds found in B.
multiplex, B. tuldoides, B. vulgaris, D. sublaevigata, and G. levis were caffeine (Figure 7), pa-
paverine (Figure 8), and sparteine (Figure 9) according to their significant peaks. Through
mass spectrometry, caffeine was found in B. multiplex, whereas papaverine was found in
B. tuldoides, B. vulgaris, D. sublaevigata, and G. levis. As for sparteine, it was found only in
B. tuldoides. Nevertheless, S. brachycladum showed no significant peaks attributed to alka-
loid compounds.
Previous studies have shown that caffeine and papaverine could influence antioxi-
dant activities. In recent decades, many scientific studies and reviews have documented
the interest in caffeine and other coffee bean constituents for their health-promoting prop-
erties [54,55]. Many authors have claimed that caffeine is a good antioxidant [56–58]. Ren
et al. [59] also reported that alkaloids in Pleioblastus amarus bamboo shoots were found to
contain caffeine when detected through ultra-high-performance liquid chromatography
(UHPLC). However, limited research has been conducted on papaverine and its links to
antioxidant properties. Interestingly, based on the study by Solmaz et al. [60], in a rat
model of sepsis-induced critical illness neuropathy, papaverine exhibited neuroprotective
effects due to its anti-inflammatory and antioxidant characteristics. The study suggested
that papaverine can contribute to antioxidant properties.
Figure 6. LC-MS/MS chromatogram of S. brachycladum.
Table 10. The compounds identified in S. brachycladum.
Peak RT (min) Identified
Compounds Molecular Formula Molecular
Weight m/z
3 0.5 Unidentified – 155.0348 156.0421
7 2.5 Amphetamine C9H13 N 113.0840 114.0913
10 5.7 Naloxone C19H21NO4327.2523 328.2596
11 7.6 Unidentified – 452.3367 453.3440
15 25.0 Perazine C20H25N3S 678.5043 340.2594
18 27.7 Unidentified – 829.5351 415.7748
24 37.4 Cyproheptadine C21 H21N 287.2828 288.2901
25 41.3 Unidentified – 315.3141 316.3214
27 77.5 Difenoconazole C19 H17Cl2N3O3309.3037 310.3110
32 78.0 RP-8-pCPT-cGMPS C16H14 ClN5O6PS2Na 311.3194 312.3267
35 78.0 Felodipine C18H19 Cl2NO4337.3365 338.3437
47 79.5 Felodipine C18H19 Cl2NO4337.3351 338.3423
52 81.4 Unidentified – 343.2726 344.2799
In brief, on the basis of the notable differences, the alkaloid compounds found in
B. multiplex,B. tuldoides,B. vulgaris,D. sublaevigata, and G. levis were caffeine (Figure 7),
papaverine (Figure 8), and sparteine (Figure 9) according to their significant peaks. Through
mass spectrometry, caffeine was found in B. multiplex, whereas papaverine was found in
B. tuldoides
,B. vulgaris,D. sublaevigata, and G. levis. As for sparteine, it was found only
in
B. tuldoides
. Nevertheless, S. brachycladum showed no significant peaks attributed to
alkaloid compounds.
Molecules 2022, 27, x FOR PEER REVIEW 14 of 24
Sparteine is a heterobicyclononane alkaloid with antiarrhythmic properties, which
can reduce the incidence of fibrillation and ventricular tachycardia, as well as help regu-
late blood pressure and heart rate [61]. Additionally, it produces a hypoglycaemic effect
and promotes the pancreatic secretion of insulin and glucagon [62]. This alkaloid has also
been linked to anti-inflammatory, antimicrobial, diuretic, and uterine contraction-induc-
ing properties [63,64]. Nonetheless, prior research indicated that sparteine lacks antioxi-
dant capabilities.
Figure 7. Chemical structures of caffeine.
Figure 8. Chemical structures of papaverine.
Figure 9. Chemical structures of sparteine.
Non-alkaloids (pharmaceutical compounds) are highlighted in Table 11. Previous
studies on biological activities have shown that anticonvulsant drugs contain loprazolam
[65], phenytoin [66], and thiopental [67] compounds. Moreover, amphetamine [68], nalox-
one [69], and perazine [70] have been found in antidepressant drugs. Antifungal drugs
also contain difenoconazole [71], econazole [72], and penconazole [73] compounds. Bio-
logical activities in antihistamine, antihypertensive, anti-inflammatory, and antipsychotic
drugs have been associated with cyproheptadine [74], felodipine [75], L-histidine [76], and
pimozide [77] compounds. Stimulant laxatives, antimicrobials, anaesthetic drugs, and dye
Figure 7. Chemical structures of caffeine.
Molecules 2022,27, 6458 14 of 23
Molecules 2022, 27, x FOR PEER REVIEW 14 of 24
Sparteine is a heterobicyclononane alkaloid with antiarrhythmic properties, which
can reduce the incidence of fibrillation and ventricular tachycardia, as well as help regu-
late blood pressure and heart rate [61]. Additionally, it produces a hypoglycaemic effect
and promotes the pancreatic secretion of insulin and glucagon [62]. This alkaloid has also
been linked to anti-inflammatory, antimicrobial, diuretic, and uterine contraction-induc-
ing properties [63,64]. Nonetheless, prior research indicated that sparteine lacks antioxi-
dant capabilities.
Figure 7. Chemical structures of caffeine.
Figure 8. Chemical structures of papaverine.
Figure 9. Chemical structures of sparteine.
Non-alkaloids (pharmaceutical compounds) are highlighted in Table 11. Previous
studies on biological activities have shown that anticonvulsant drugs contain loprazolam
[65], phenytoin [66], and thiopental [67] compounds. Moreover, amphetamine [68], nalox-
one [69], and perazine [70] have been found in antidepressant drugs. Antifungal drugs
also contain difenoconazole [71], econazole [72], and penconazole [73] compounds. Bio-
logical activities in antihistamine, antihypertensive, anti-inflammatory, and antipsychotic
drugs have been associated with cyproheptadine [74], felodipine [75], L-histidine [76], and
pimozide [77] compounds. Stimulant laxatives, antimicrobials, anaesthetic drugs, and dye
Figure 8. Chemical structures of papaverine.
Molecules 2022, 27, x FOR PEER REVIEW 14 of 24
Sparteine is a heterobicyclononane alkaloid with antiarrhythmic properties, which
can reduce the incidence of fibrillation and ventricular tachycardia, as well as help regu-
late blood pressure and heart rate [61]. Additionally, it produces a hypoglycaemic effect
and promotes the pancreatic secretion of insulin and glucagon [62]. This alkaloid has also
been linked to anti-inflammatory, antimicrobial, diuretic, and uterine contraction-induc-
ing properties [63,64]. Nonetheless, prior research indicated that sparteine lacks antioxi-
dant capabilities.
Figure 7. Chemical structures of caffeine.
Figure 8. Chemical structures of papaverine.
Figure 9. Chemical structures of sparteine.
Non-alkaloids (pharmaceutical compounds) are highlighted in Table 11. Previous
studies on biological activities have shown that anticonvulsant drugs contain loprazolam
[65], phenytoin [66], and thiopental [67] compounds. Moreover, amphetamine [68], nalox-
one [69], and perazine [70] have been found in antidepressant drugs. Antifungal drugs
also contain difenoconazole [71], econazole [72], and penconazole [73] compounds. Bio-
logical activities in antihistamine, antihypertensive, anti-inflammatory, and antipsychotic
drugs have been associated with cyproheptadine [74], felodipine [75], L-histidine [76], and
pimozide [77] compounds. Stimulant laxatives, antimicrobials, anaesthetic drugs, and dye
Figure 9. Chemical structures of sparteine.
Previous studies have shown that caffeine and papaverine could influence antioxi-
dant activities. In recent decades, many scientific studies and reviews have documented
the interest in caffeine and other coffee bean constituents for their health-promoting
properties [54,55]
. Many authors have claimed that caffeine is a good
antioxidant [56–58]
.
Ren et al. [
59
] also reported that alkaloids in Pleioblastus amarus bamboo shoots were found
to contain caffeine when detected through ultra-high-performance liquid chromatography
(UHPLC). However, limited research has been conducted on papaverine and its links to
antioxidant properties. Interestingly, based on the study by Solmaz et al. [
60
], in a rat model
of sepsis-induced critical illness neuropathy, papaverine exhibited neuroprotective effects
due to its anti-inflammatory and antioxidant characteristics. The study suggested that
papaverine can contribute to antioxidant properties.
Sparteine is a heterobicyclononane alkaloid with antiarrhythmic properties, which
can reduce the incidence of fibrillation and ventricular tachycardia, as well as help regulate
blood pressure and heart rate [
61
]. Additionally, it produces a hypoglycaemic effect
and promotes the pancreatic secretion of insulin and glucagon [
62
]. This alkaloid has
also been linked to anti-inflammatory, antimicrobial, diuretic, and uterine contraction-
inducing properties [
63
,
64
]. Nonetheless, prior research indicated that sparteine lacks
antioxidant capabilities.
Non-alkaloids (pharmaceutical compounds) are highlighted in Table 11. Previous stud-
ies on biological activities have shown that anticonvulsant drugs contain
loprazolam [65]
,
phenytoin [
66
], and thiopental [
67
] compounds. Moreover, amphetamine [
68
],
naloxone [69]
,
and perazine [
70
] have been found in antidepressant drugs. Antifungal drugs also contain
difenoconazole [
71
], econazole [
72
], and penconazole [
73
] compounds. Biological activities
in antihistamine, antihypertensive, anti-inflammatory, and antipsychotic drugs have been
associated with cyproheptadine [
74
], felodipine [
75
], L-histidine [
76
], and pimozide [
77
]
compounds. Stimulant laxatives, antimicrobials, anaesthetic drugs, and dye agents contain
bisacodyl [
78
], phytosphingosine [
79
], cinchocaine [
80
], and pararosaniline [
81
] compounds.