SAJB-02536; No of Pages 5

South African Journal of Botany xxx (2019) xxx

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South African Journal of Botany

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Leaf harvesting severity affects total phenolic and tannin content of fresh
and dry leaves of Moringa oleifera Lam. trees growing in Gauteng,
South Africa
E.S. du Toit ⁎, J. Sithole, J. Vorster
Department of Plant and Soil Sciences, Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria 0002, South Africa

a r t i c l e i n f o a b s t r a c t

Article history: The most important chemicals common in Moringa oleifera Lam. leaves are polyphenols and tannins. They are
Received 10 May 2019 synthesised during development and the amount and composition are primarily dependent on environmental
Received in revised form 22 July 2019 conditions and factors such as leaf harvesting. Trials were conducted in the Gauteng Province at the University
Accepted 20 August 2019
of Pretoria Experimental Farm on Hillcrest Campus (250 45′ 08.6″S, 280 15′ 30.S" E), with an altitude of
Available online xxxx
1372 m above sea level and an average annual rainfall of 640 mm. Five year old M. oleifera tree orchard were
Edited by AR Ndhlala used for this study and treatments were laid in a randomised complete block design, where 12 plants were
used in each plot within a row, with a plant spacing of 2 × 2 m2. Partial harvesting was done at 25% leaf removal,
Keywords: while severe leaf harvesting was done at 75% leaf removal. The control trees were not harvested, but only leaf
Air drying (mature and immature) samples were harvested for analyses. Leaf samples from all plots were harvested in Sep-
Metabolites tember, November, February and May in years 1 and 2. Leaf samples (equal quantities of immature and mature
Moringa oleifera leaves) from partially, severely harvested and control plots were harvested and separated into batches, one to be
Partial leaf harvesting analysed as fresh material and the other as dry material. The dry material was placed in paper bags and allowed to
Severe leaf harvesting
immediately air dry under normal room conditions of 26 °C. Partially harvested M. oleifera leaves produced an
increased accumulation of total phenolic content (TPC) 35–44 g/kg dry weight, from spring to summer season,
in comparison to severe harvesting 26–35 g/kg dry weight. With severe leaf harvesting (75%), because of prob-
able lower photosynthetic capacity, phenols across September to May of years 1 and 2 decreased. The onset of
low temperatures in May resulted mostly higher TPC and tannin build up, which means that cold stress may im-
prove more bio-active compounds in the leaves. Dark and fully developed mature leaves mainly contained the
highest amount of TPC. Moringa oleifera leaves collected and dried at each harvesting interval, instead of freshly
milled, do show the same quantity of TPC and tannins across the season illustrating that air drying can be used as
a post-harvest handling technique, extending shelf life without losses of vital phenols. With harvesting severity
as a concern in Moringa, this study confirms that the TPC and tannins in mature and immature leaves did not rise
to toxic levels for human consumption.
© 2019 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction
The genus Moringa has been found to be a good source of polyphe-
nols, as the leaves of moringa have various biological activities includ-
ing, anticancer activities, prevention of cardiovascular diseases, liver
disease (Kumar and Pari, 2003), antitumor, skin disorders, inflamma-
tion, digestive disorders, skin disorders and regulation of thyroid status
(Cajuday and Pocsidiv, 2010). The most important chemicals which are
common in moringa leaves are polyphenols and tannins (Leone et al.,
2015; Moyo et al., 2012; Pakade et al., 2013). High concentrations of
tannins, which are polyphenols, cause lower feed intake, growth rate,
⁎ Corresponding author.
E-mail address: elsa.dutoit@up.ac.za (E.S. du Toit).
feed efficiency, net metabolisable energy, and protein digestibility, and
as such foods which are high in tannins are regarded to be of poor nutri-
tional value (Chung et al., 1998; Makkar, 2003;). Phenolic compounds
and tannins are synthesised by plants during growth and development
(Arena and Radice, 2016; Harborne, 1982) and are influenced by condi-
tions such as leaf age, harvesting and pruning (Nobosse et al., 2018;
Rahmani et al., 2015). Leaf harvesting is an exclusive target of moringa
production in most African countries. Although information on the ef-
fect of phenological growth flushes and stages of certain moringa spe-
cies is available (Muhl et al., 2011; Arena and Radice, 2016),
information on the effect of leaf harvesting intensity on polyphenol
and tannin accumulation in Moringa oleifera is still lacking.
Phenolic compounds are plant secondary metabolites playing a role
in plant resistance. These phenolics are one of the most diverse groups

https://doi.org/10.1016/j.sajb.2019.08.035
0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.

Please cite this article as: E.S. du Toit, J. Sithole and J. Vorster, Leaf harvesting severity affects total phenolic and tannin content of fresh and dry
leaves of Moring..., South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.08.035
2 E.S. du Toit et al. / South African Journal of Botany xxx (2019) xxx

Table 1 was placed in paper bags and allowed to air dry under normal room
Effect of leaf harvesting severity on TPC (g/kg dry weight). conditions of 26 °C. The fresh material samples were immediately
Harvesting severity Control Partial Severe placed in polythene bags and kept in cold chambers for laboratory anal-
Leaf maturity Months TPC ± SE TPC ± SE TPC ± SE
yses. The leaves were harvested on 15 September, 15 November, 15
February and 15 May precisely from the same marked points. Data
Immature leaves September 30b ± 1.05 32b ± 0.64 28ab ± 0.94
were collected consistently on the above timeline on the same trees.
November 27b ± 0.95 31bc ± 0.63 24b ± 1.34
February 31b ± 1.94 32b ± 0.65 24b ± 0.93 Before cold chamber storage and air drying, mature leaves harvested
May 37ab ± 0.55 38ab ± 1.34 26b ± 0.45 at each interval were classed as dark and fully expanded, while imma-
ture leaves were classed as light, tender and partially developed. Leaf
Mature leaves September 33b ± 0.67 35b ± 0.84 26b ± 0.58
November 27b ± 1.25 26c ± 1.45 28b ± 1.23 samples from control, partially and severely plants were then air dried
February 42a ± 1.88 44a ± 1.48 35a ± 1.11 at 26 °C for 21 days in the shade, milled using CNC (Computer Numeri-
May 47a ± 1.54 47a ± 1.28 36a ± 1.65 cally Controlled) Knee milling machine (JET JTM-1050 Mill, HIS Engi-
Values with same letter are statistically non-significant at p ≤ .05. TPC - Total Phenolic Con- neering, Japan) and then analysed for phenol and tannin content,
tent; SE - Standard Error. while the rest were kept at −15 °C storage for fresh mass analyses.
To follow leaf composition of a tree, a branch was marked using a red
tag on three identified branches on each tree and immature and mature
of plant active substances. These compounds take part in regulation of leaves were then counted. The total number of mature and immature
seed germination and cooperative in regulating the growth of plants, leaves per tree were then averaged by dividing by three and then the
also taking part in the defence responses during infection, excessive quantity of mature and immature leaves was then calculated over 100
sun exposure, injuries and heavy metal stress (Kulbat, 2016). Tannins to estimate total percentage.
are water-soluble polyphenols protecting plants from herbivores
(Chung et al., 1998). The objective of this study was to investigate the 2.2. Chlorophyll content
effect of leaf harvesting severity of Moringa oleifera on total phenolic
content and tannin accumulation. At 7-day intervals, a Minolta SPAD-502 chlorophyll meter (Minolta
Camera Co., Ltd., Japan) was used to measure the chlorophyll content
2. Materials and methods of the M. oleifera leaves. Chlorophyll meter readings were taken be-
tween the midrib and the leaf margin from leaflets, which were ran-
2.1. Leaf harvesting treatments domly selected from the youngest leaf on trees within each treatment
at around 12:00 noon in the afternoon for consistency.
The trials were conducted in the Gauteng province of South Africa,
on the University of Pretoria Experimental Farm at Hillcrest Campus 2.3. Extraction and measurement of total phenolic content (TPC) and
(25°45′S, 28°16′E), with an altitude of 1372 m above sea level and an tannins
average annual rainfall of 674 mm. Five year old Moringa oleifera trees
were used for this study and treatments were laid in a Randomised A tannin extract was prepared by adding 100 mg sample of milled M.
Complete Block Design where 12 plants were used in each plot within oleifera leaves into a 15 ml plastic tube with 5 ml acetone according to
a row and with a plant spacing of 2 × 2 m2. General orchard manage- the method of Makkar (2003). Each sample was vortexed and placed
ment practices, such as weed and pesticide management (if occurred) into a water bath at 25 °C for 20 min. The sample was then centrifuged
were done across all treatments during the trial period. To adjust nitro- at low speed for 10 min and the supernatant was then collected. The
gen deficiencies evident from soil analysis results, 20 g of Limestone procedure was repeated to collect another supernatant. Aliquots of tan-
Ammonia Nitrate (LAN) (28) fertiliser was applied to each tree, nin containing extract (initially 0.02, 0.05 and 0.1 ml) for control, par-
16 weeks after trial commencement. Trees were single stemmed with tially and severely harvested samples were taken and placed into test
a mean height of 254 cm and width circumference of 13.5 cm. Partial tubes, and the volume was made up to 0.5 ml with distilled water.
harvesting was done at 25% leaf removal, while 75% of leaf removal Then 0.25 ml of the Folin–Ciocalteu reagent and 1.25 ml of the sodium
was done for severe leaf harvesting. Leaves were harvested from carbonate solution of pH 10.66 (Merck, South Africa) were added.
below to the upper parts of the canopy. From the control trees immature Tubes were vortexed and absorbance was recorded at 725 nm after
and mature leaves were only sampled for analyses. Leaf samples (equal 40 min and the total phenolic content (TPC) was calculated from the
quantities of immature and mature leaves) from partially, severely har- harvested leaf samples as tannic acid equivalents from the calibration
vested and control plots were harvested and separated into batches, one curve. Then, 100 mg of polyvinylpolypyrrolidone (PVPP) (sufficient to
to be analysed as fresh material and the other as dry. The dry material bind 2 mg of total phenols) was weighed and put into 100 × 12 mm

50
45 a a
a a a b
40 a b b
Measured SPAD value

b
(Chlorophyll content)

35 a Partial
b
30
Severe
25
20 Control
15
10
5
0
September November February May
Months

Fig. 1. Leaf chlorophyll content in mature leaves of Moringa oleifera, measured on trees under different leaf harvesting intensity treatments. Values with same letter are statistically
non-significant at p ≤ .05.

Please cite this article as: E.S. du Toit, J. Sithole and J. Vorster, Leaf harvesting severity affects total phenolic and tannin content of fresh and dry
leaves of Moring..., South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.08.035
 E.S. du Toit et al. / South African Journal of Botany xxx (2019) xxx 3

Table 2 than the latter. This concurs with work done by Iqbal and Bhanger
Effect of leaf harvesting intensity on tannin content (g/kg dry weight). (2005) who highlighted that TPC can be a function of leaf harvesting
Harvesting severity Control ± SE Partial ± SE Severe ± SE treatment.
Leaf maturity Months
Previous work by Moyo et al. (2012), Sharmin et al. (2011) and
Pakade et al. (2013) on acceptable TPC content for animal and human
Immature leaves September 26a ± 1.23 25a ± 1.28 31ab ± 0.64
consumption indicates that quantities obtained in this study were
November 28ab ± 1.89 27a ± 1.56 29a ± 0.57
February 27ab ± 0.83 28ab ± 0.94 34a ± 1.45 within acceptable ranges. Chlorophyll content build-up in partially har-
May 32ab ± 0.74 31ab ± 0.56 36a ± 1.54 vested plants across months, as shown in Fig. 1, may help explain in-
creased TPC across months. Generally chlorophyll content as well as
Mature leaves September 16b ± 1.22 17b ± 0.55 25a ± 1.33
November 23b ± 1.09 18b ± 1.44 31a ± 1.46 TPC increased in February (summer), indicating that a positive relation-
February 26a ± 1.76 19b ± 1.64 31a ± 1.76 ship exists between chlorophyll content and TPC. Chlorophyll content
May 36a ± 1.34 23b ± 1.34 38a ± 0.92 may influence TPC through photosynthesis (Abdulkadir et al., 2015).
Values with same letter are statistically non-significant at p ≤ .05. SE - Standard Error.
3.2. Total tannin content

test tubes. Finally, 1 ml of distilled water and 1 ml of tannin-containing
extract were added. The mixture from each tube was then vortexed and
centrifuged (3000 g for 10 min) and the supernatant was collected and
absorbance was recorded at 725 nm. The total tannins were calculated
as follows: Total tannins = Total phenols − Non-tannin phenols.
Data were then statistically analysed using the Statistical Analysis
Software (SAS version 9.2) program for Microsoft Windows. Analysis
of Variance (ANOVA General Linear Model) was used to assess if differ-
ences between treatments were significantly different (p ≤ .05) (Blom,
1958).
3. Results and discussion
Values reported were averaged for the two seasons.
3.1. Total phenolic content (TPC)
In general, TPC increased from spring (September) to winter (May)
in the control and partially harvested trees from immature leaves and
in all treatments from mature leaves. Immature leaf samples from se-
vere leaf harvesting recorded significantly (p ≤ .05) lower TPC than sam-
ples from the control and partial leaf harvesting from November until
May. Mature leaf samples from severe leaf harvesting were significantly
(p ≤ .05) lower than the control and partial leaf harvesting from Febru-
ary to May (Table 1). Table 1 illustrates that mature leaves have more
TPC than immature leaves, as recorded during February and May period.
Chlorophyll content measured on mature leaves in February (Fig. 1)
do show that partial leaf harvesting recorded higher chlorophyll content
in leaves on the trees than severe leaf harvesting, explaining possible
higher photosynthetic activity and therefore higher TPC in the former
Immature leaves of severely harvested trees had higher tannin con-
tent across all months than the other treatments (Table 2). The tannin
content in mature leaves of severely harvested trees were significantly
(p ≤ .05) higher than those from partial leaf harvesting throughout the
season and for the control in September and November (Table 2). Tan-
nins generally increased as the season progressed across all treatments.
Lower temperatures experienced towards May (winter) could have
contributed to tannin build-up as the trees were initiating ways of de-
fence against cold stress (Iqbal and Bhanger, 2006).
Higher tannin accumulation on severely harvested leaves could also
be attributed to the defence mechanisms the plant acquired in trying to
protect itself against excessive leaf loss. Tannins are considered impor-
tant quantitative protective compounds conferring defence against her-
bivory and stress (Chung et al., 1998; Rhodes and Cates, 1976), however
high contents are not desirable for human consumption (Chung et al.,
1998). High levels of tannin ingestion by animals can result in toxicity
and can lead to death (Makkar, 2003). Even though consumption of
fully developed and mature leaves does not usually lead to death of an-
imals, their consumption impede production. However, the tannin con-
tent recorded in this study, would not produce any adverse effects when
eaten by animals (Makkar, 2003).
Previous research has found a relationship between canopy struc-
ture and sunlight exposure and subsequent plant compounds
(Rahmani et al., 2015). Plants are potential sources of natural bioactive
compounds such as secondary metabolites. They absorb the sunlight
and produce high levels of oxygen and the above metabolites via photo-
synthesis. Flavonoids and phenolics are important groups of secondary
metabolites and bioactive compounds in plants (Kim et al., 2003).
Phenolics and flavonoid syntheses in plants are influenced by envi-
ronmental factors. Light is one of the most important environmental

Table 3
Summary on influence of leaf age on TPC and tannin content (g/kg dry weight). TPC - Total Phenolic Content.

Immature Mature

Harvesting severity Months TPC ± SE Tannin ± SE Average TPC ± SE Tannin ± SE Average

September 30 ± 0.89 26 ± 0.45 28 33 ± 0.34 16 ± 0.35 25

Control November 27 ± 1.23 28 ± 1.24 28 27 ± 1.34 23 ± 1.54 25
February 31 ± 0.56 27 ± 1.45 29 42 ± 0.64 26 ± 0.54 34
May 37 ± 1.32 32 ± 1.89 35 48 ± 1.46 36 ± 0.46 42
Average 32ab 28a 30a 38a 25ba 32a

September 32 ± 0.24 25 ± 1.34 32 35 ± 1.34 17 ± 1.44 26

Partial November 31 ± 0.45 27 ± 0.64 29 26 ± 0.56 18 ± 0.76 22
February 32 ± 0.33 28 ± 0.94 30 44 ± 1.23 19 ± 0.98 32
May 38 ± 1.35 31 ± 1.34 35 47 ± 1.45 23 ± 1.23 35
Average 33ab 28a 32a 38a 19b 29ab

September 28 ± 1.34 31 ± 0.76 30 26 ± 1.34 25 ± 1.76 26

Severe November 24 ± 0.46 29 ± 0.64 27 28 ± 1.33 31 ± 1.94 30
February 24 ± 0.74 34 ± 1.23 29 35 ± 1.23 31 ± 1.38 33
May 26 ± 0.48 36 ± 1.44 31 36 ± 0.45 38 ± 1.38 37
Average 26b 33b 29b 31b 31ab 32ab

Values with same letter are statistically non-significant at p ≤ .05. SE - Standard Error.

Please cite this article as: E.S. du Toit, J. Sithole and J. Vorster, Leaf harvesting severity affects total phenolic and tannin content of fresh and dry
leaves of Moring..., South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.08.035
4 E.S. du Toit et al. / South African Journal of Botany xxx (2019) xxx

Table 4
Average leaf percentage harvested over time for two years.

Leaf harvesting severity Average Leaf % Sept ± SE Nov ± SE Feb ± SE May ± SE

Partial harvesting Immature 65a ± 0.34 55a ± 1.65 41c ± 0.81 38c ± 1.43
Mature 35b ± 1.54 45c ± 1.45 59b ± 1.45 62b ± 0.45

Severe harvesting Immature 73a ± 0.56 76a ± 1.83 83a ± 1.65 85a ± 1.55
Mature 27b ± 1.56 24c ± 0.89 17d ± 1.45 15d ± 0.34

Values with same letter are statistically non-significant at p ≤ .05. SE - Standard Error.

stimuli, not only involved in the regulation of plant growth and organo-
genesis, but also in the biosynthesis of plant products including both
primary and secondary metabolites (Ghasemzadeh et al., 2010). Pheno-
lic biosynthesis requires light or is enhanced by light, and flavonoid for-
mation is absolutely light dependent and its biosynthetic rate is related
to light intensity and density (Xie and Wang, 2006). It was revealed that
changes in light intensity with changes in plant morphology and phys-
iological characteristics were able to change the production of tannins
and total phenolic compounds in plants (Ghasemzadeh et al., 2010).
In this study, severe harvesting increased tannins considerably, while
partial harvesting improved TPC, suggesting that leaf harvesting of
about 25% is ideal in improving TPC, and as the intensity increased to
about 75% (severe) the plant signalled this as stress and as such it in-
creased manufacturing of defence metabolites hence higher tannin
levels were recorded.
The difference in TPC and tannin compounds in leaves (mature and
immature) was probably due to the leaf harvesting treatments which
were affected by sunlight penetration and as a result the trees ability
to photosynthesize optimally. Phenolic compounds are synthesised
from the phenylalanine produced by the shikimic acid pathway. The de-
amination of phenylalanine catalysed by enzyme phenylalanine
ammonia-lyase (PAL) is the first step in this biosynthetic pathway.
PAL is induced by a variety of stimuli such as light irradiation (Ruiz-
Garcia and Gomez-Plaza, 2013).
Observations from Julkunen-Tiitto (1989) and Wiermann (1981)
suggest that TPC increases with an increase in the age of leaves and is
minimal in early stages of leaf development and growth, but moderately
increase when leaves mature, which we can concur with our findings.
Work done by Sreelatha and Padma (2009) on the other hand confirms
that TPC vary with the stage of maturity in Moringa leaves. Table 4
shows an increase in mature over immature leaf percentages for par-
tially harvested trees, suggesting that trees recovered with quantitative
increase in mature leaves as the season progressed. The highest amount
of TPC and tannins were recorded in May, while the lowest were re-
corded in November (Table 3). This concurs with reports by Wang
and Zheng (2001) demonstrating that temperature greatly modifies
phenolic compound properties.
With severely harvested trees, there was a decline in mature leaves,
and immature leaves increased immensely from September to May.
These trees recovered slower from the harvesting as leaves may not
have reached maturity after May. Conversely, with partial harvesting,
immature leaves decreased over time while mature leaves increased
as shown in Table 4.
Reports from by Hyder et al. (2002) reveal that immature leaves of a
plant contain high levels of tannins in comparison to mature leaves, es-
pecially during stress conditions. This concurs with observations re-
corded on our harvesting treatments, where immature leaves had
slightly more tannins, especially from the partially harvested trees, but
also from the severely treated trees.

3.3. Summary on influence of leaf age on TPC and tannin content 3.4. Post-harvest handling effect on TPC and tannin content

Generally mature leaves had higher TPC than immature leaves, spe-
cifically significant (p ≤ .05) in the control and partially harvested trees
in February and May. Immature leaves were predominantly higher in
tannin content than mature leaves, distinctively significant (p ≤ .05)
on partially harvested trees.
Table 5
Effect of postharvest handling on TPC and tannin content (g/kg dry weight).
Fresh Dry
Tannin and TPC increased across the season in both fresh and dry
samples (Table 5). No significant (p ≤ .05) TPC difference was recorded
between dry and fresh samples across the season.
This confirms studies by Yang et al. (2006) that air drying as a treat-
ment on Moringa does not change the phytochemical content of leaves.
It could be that drying temperatures and conditions were not enough to
cause interconversions between phenolic molecules, although prior re-
ports suggest that drying may decrease total phenolic content (Deepa
et al., 2007).

Leaf harvesting Months TPC Tannin Average TPC Tannin Average 4. Conclusions
severity

September 23 25 24 25 23 24
Partial leaf harvesting was the ideal harvesting practise to maximise
Control November 25 25 25 25 23 24 total phenolic content in mature leaves coupled with lower tannin con-
February 31 22 26 28 18 23 tent in comparison to severe leaf harvesting and the control. The onset
May 34 28 31 32 21 26 of low temperatures in May resulted mostly higher TPC and tannin
Average 28a 25a 27a 28a 21a 24a
build up which means that cold stress may improve more bio-active
September 25 23 24 26 22 24 compounds in the leaves. Dark and fully developed mature leaves
Partial November 26 29 28 26 25 26 mainly contained the highest amount of TPC. Severe leaf harvesting in-
February 35 33 34 29 28 29
creased tannin build-up, especially in immature leaves. With severely
May 36 31 34 35 29 32
Average 30a 29a 30a 29a 26a 28a harvested trees, there was a decline in mature leaves and immature
leaves increased immensely on these trees as the season progress.
September 26 23 25 24 22 23
These trees recovered slower from the harvesting as leaves could not
Severe November 24 26 25 22 24 23
February 25 33 29 23 31 28 reach maturity before the winter.
May 23 34 28 21 31 26 Air drying does not decrease tannin levels in leaves as a means of
Average 25a 29a 27a 23a 27a 25a post-harvest treatment, nor does it alter the total phenolic content in
Values with same letter are statistically non-significant at p ≤ .05. TPC - Total Phenolic leaves. From an agronomic point of view, it means farmers can employ
Content. cheap and sustainable post-harvest control by air drying (under room

Please cite this article as: E.S. du Toit, J. Sithole and J. Vorster, Leaf harvesting severity affects total phenolic and tannin content of fresh and dry
leaves of Moring..., South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.08.035
 E.S. du Toit et al. / South African Journal of Botany xxx (2019) xxx
temperature in a shade) their Moringa leaf produce, without altering the
total phenolic content. Our study suggests that M. oleifera leaves can be
consumed at any time of the year or when feed is scarce, and it can be
transported to areas where it is not cultivated without any loss of leaf
quality.
Acknowledgements
We thank University of Pretoria, South Africa and National Research
Foundation (NRF) Competitive programme for rated researchers - Prof
Elsa S. du Toit (Ref: CPRR14072981925), for facilities and funding.
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