plants
Article
Mineral Fertilization Influences the Growth, Cryptolepine
Yield, and Bioefficacy of Cryptolepis sanguinolenta
(Lindl.) Schlt.
Jacqueline Naalamle Amissah 1,* , Forgive Enyonam Alorvor 1, Benjamin Azu Okorley 1 , Chris Mpere Asare 2,
Dorcas Osei-Safo 3 , Regina Appiah-Opong 4 and Ivan Addae-Mensah 3
1 Crop Science Department, University of Ghana, Legon, Accra P.O. Box LG 44, GM, Ghana;
pe4give@gmail.com (F.E.A.); bazu_okorley@st.ug.edu.gh (B.A.O.)
2 Council for Scientific and Industrial Research (CSIR), Plant Genetic Resources Research Institute (PGRRI),
Bunso P.O. Box 7, EE, Ghana; chrisasare83@gmail.com
3 Chemistry Department, University of Ghana, Legon, Accra P.O. Box LG 56, GM, Ghana;
dosei-safo@ug.edu.gh (D.O.-S.); iaddae-mensah@ug.edu.gh (I.A.-M.)
4 Department of Clinical Pathology, Noguchi Memorial Institute for Medical Research, College of Health
Sciences, University of Ghana, Legon, Accra P.O. Box LG 581, GM, Ghana;
rappiah-opong@noguchi.ug.edu.gh
* Correspondence: jnamissah@ug.edu.gh
Abstract: Cryptolepis sanguinolenta (Lindl.) Schlt., the main source of cryptolepine alkaloid, is
intensively exploited in the wild to treat malaria and Lyme disease. In this study, the influence of
four inorganic fertilizers (supplying N, P, K, or NPK) and four growth periods (3, 6, 9, and 12 months





 after transplanting) on the herb’s root biomass, cryptolepine content and yield, and biological
activities were investigated in a pot and field trial. The results showed the application of N (in the
Citation: Amissah, J.N.; Alorvor, F.E.;
form of Urea or NPK) increased root biomass yield, cryptolepine content, and cryptolepine yield
Okorley, B.A.; Asare, C.M.; Osei-Safo,
compared to unfertilized plants. The 9-month-old plants recorded the maximum cryptolepine content
D.; Appiah-Opong, R.;
Addae-Mensah, I. Mineral (2.26 mg/100 mg dry root) and cryptolepine yield (304.08 mg/plant), indicating the perfect time to
Fertilization Influences the Growth, harvest the herb. Plant age at harvest had a more significant influence (50.6–55.7%) on cryptolepine
Cryptolepine Yield, and Bioefficacy of production than fertilizer application (29.2–33.3%). Cryptolepine extracts from 9- to 12-month-old
Cryptolepis sanguinolenta (Lindl.) plants had the highest antiplasmodial activity (IC50 = 2.56–4.65 µg/mL) and drug selectivity index
Schlt.. Plants 2022, 11, 122. https:// (2.15–3.91) against Plasmodium falciparum Dd2. These extracts were also cytotoxic to Jurkat leukaemia
doi.org/10.3390/plants11010122 cell lines (CC50 < 62.56 µg/mL), indicating the possible use of cryptolepine for cancer management.
Academic Editor: Domenico Growing the herb in the field increased cryptolepine yield 2.5 times compared to growth in a pot,
Trombetta but this did not influence the antiplasmodial activity of the extract. Commercial cultivation of
C. sanguinolenta for 9 months combined with N application could be a promising solution to the
Received: 28 October 2021
sustainable use of this threatened medicinal species.
Accepted: 23 November 2021
Published: 1 January 2022
Keywords: antiplasmodial; cytotoxicity; domestication; endangered medicinal plant; growth pe-
Publisher’s Note: MDPI stays neutral riod; malaria
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Cryptolepis sanguinolenta (Lindl.) Schlecter (family Periplocaceae) is among the indige-
nous herbal vines that have received much attention in West African traditional medicine.
Copyright: © 2022 by the authors. Its pharmacological activities are due to the predominant alkaloid cryptolepine along with
Licensee MDPI, Basel, Switzerland. the minor alkaloids (cryptoquindoline, quindoline, biscryptolepine, and cryptospirolepine)
This article is an open access article
contained in the root of the plant [1–4]. In Ghana, C. sanguinolenta is widely used in
distributed under the terms and
decoctions and powders to treat malaria, a deadly disease of concern for public health,
conditions of the Creative Commons
Attribution (CC BY) license (https:// especially in areas where orthodox medicines are not readily available [5,6]. Currently, it is
creativecommons.org/licenses/by/ being studied in clinical trials as a potential therapy for the novel coronavirus disease 2019
4.0/). (COVID-19) [7,8]. Elsewhere in the USA, the herb is used to treat Babesia, Lyme disease
Plants 2022, 11, 122. https://doi.org/10.3390/plants11010122 https://www.mdpi.com/journal/plants
Plants 2022, 11, 122 2 of 16
(Borreliosis burgdorferi), and Bartonella [9]. It is also appreciated for its antibacterial [10],
anticancer [11,12], anti-diabetic [13], antifungal [14], and anti-inflammatory properties [15].
In recent years, Ghana’s herbal and brewery industries have seen an increase in de-
mand for commercial products, including herbal teas, Class Malacure, Herbaquine, Malaherb,
and Phyto-Laria, made from the roots of C. sanguinolenta. This demand has resulted in
an excessive indulgence in harvesting from the wild, especially during the flowering pe-
riod of the plant. This situation, coupled with climate change and land degradation, has
drawn the attention of both ecologists and plant growers to the importance of protecting
C. sanguinolenta from extinction [16]. There is an urgent need to champion domestica-
tion and cultivation protocols for the production of C. sanguinolenta to conserve the wild
population and provide a sustainable source of plant material for use.
Earlier studies by Amissah et al. [17] found that the staking of C. sanguinolenta plants
promoted pod formation and subsequent seed development. Additionally, protocols
for the micropropagation of C. sanguinolenta have been developed to multiply planting
materials [18]. However, the effects of environmental factors (for example, nutrient, climate,
and water requirements) and the absence of reproducible bioactivity represent a major
impediment in the cultivation of most medicinal plants [19,20]. Among these factors,
plant nutrition has the greatest impact on plant growth and secondary metabolite yield,
especially in marginal soils [21–23]. In recognition of the important role of plant nutrition,
several studies have documented the nutritional requirements of other industrial and
medicinal plants [23]. However, the nutrient demands and precise fertility targets of C.
sanguinolenta as a commercial crop are unknown.
Furthermore, the economic value of C. sanguinolenta is determined by the cryptolepine
content in the roots. Consequently, any rise in the cryptolepine levels provides a signif-
icant economic gain. Studies on Periwinkle (Vinca minor) and Datura (Datura innoxia)
reported a rise in alkaloid content following the application of mineral NPK and N fertil-
izer, respectively [24,25]. Additionally, the use of fertilizers, notably those that provide
nitrogen (N) and phosphorus (P), has been reported to improve the biomass of vegetative
parts that produce secondary metabolites in aromatic medicinal plants (e.g., Foeniculum
vulgare—fennel, Cymbopogon martinii—palmarosa, and Ocimum basilicum—basil culti-
vars) [26–28]. Therefore, the supply of mineral nutrients (N, P, or K) is expected to influence
the production and accumulation of cryptolepine in C. sanguinolenta.
Amid efforts toward the cultivation and conservation of C. sanguinolenta in Ghana, the
present study reports the comparative effects of four inorganic fertilizers (supplying either
N, P, K, or combined NPK) and plant age on plant biomass accumulation, cryptolepine
content, and yield. The bioactivity of cryptolepine extracts from plants of different ages
was also determined on Plasmodium parasite and cancer cells. Such information will
ultimately help understand the effect of inorganic fertilizers and plant growth periods on
the commercial production of C. sanguinolenta.
2. Results and Discussion
2.1. Effect of Fertilizer Application on Vegetative Growth and Biomass Yield of C. sanguinolenta
To develop C. sanguinolenta into a profitable commercial crop, its agronomic perfor-
mance needs to be improved. Therefore, in this study, the effect of four mineral fertilizers
and four growth periods on the growth of C. sanguinolenta were investigated to provide
information on the nutrients required to enhance its cultivation and productivity.
The data presented in Table 1 reveal that fertilizer treatments significantly influenced
the vine number and girth in both the field and pot experiments. First, fertilizer P at
250 kg/ha and NPK at 262.5 kg/ha gave the highest number of vines (≈5) compared to the
unfertilized controls in the field experiment. Additionally, all NPK treatments, except for
NPK at 225 kg/ha, yielded the thickest vines for 3-, 6- and 12-month-old plants (3.97, 4.50,
and 5.15 mm, respectively). Taking time into account, the application of NPK at 187.5 kg/ha
alongside the 6-month growth period was the most effective for increasing vine girth and
number, as these parameters in both experiments were consistently increased.
Plants 2022, 11, 122 3 of 16
Table 1. Effects of inorganic fertilizers on vine number and girth of C. sanguinolenta at different plant
growth periods.
Treatment Field Experiment Pot Experiment
Fertilizer Plant Age
(kg/ha) (in MAT) No of Vines Vine Girth (cm) No of Vines Vine Girth (cm)
CK 3 2.33 ± 0.33 a 2.90 ± 0.35 abc 2.33 ± 0.33 ab 1.47 ± 0.46 a
K at 150 3 3.33 ± 0.33 abc 3.27 ± 0.29 bcd 2.33 ± 0.33 ab 1.57 ± 0.37 a
N at 100 3 3.67 ± 0.33 abcd 2.49 ± 0.33 ab 3.00 ± 0.58 ab 2.07 ± 0.34 abc
NPK at 150 3 4.33 ± 0.33 bcd 3.97 ± 0.17 cde 3.67 ± 0.33 bc 2.20 ± 0.58 abc
P at 100 3 3.00 ± 0.12 ab 3.23 ± 0.88 bcd 3.33 ± 0.33 abc 1.63 ± 0.34 ab
CK 6 3.00 ± 0.12 ab 2.63 ± 0.36 abc 3.67 ± 0.33 bc 2.04 ± 0.09 abc
K at 187.5 6 3.33 ± 0.33 abc 3.54 ± 0.58 bcd 4.67 ± 0.33 cd 3.79 ± 0.58 de
N at 150 6 4.67 ± 0.33 bcd 3.64 ± 0.37 bcd 5.33 ± 0.33 d 3.73 ± 0.58 de
NPK at 187.5 6 5.00 ± 0.17 cd 4.50 ± 0.66 de 7.00 ± 0.58 e 3.70 ± 0.58 de
P at 150 6 3.67 ± 0.33 abcd 2.82 ± 0.29 abc 6.00 ± 0.58 de 2.81 ± 0.58 abcde
CK 9 3.67 ± 0.33 abcd 2.70 ± 0.11 abc 3.33 ± 0.33 abc 2.69 ± 0.46 abcde
K at 225 9 3.67 ± 0.33 abcd 2.63 ± 0.17 abc 4.67 ± 0.88 cd 2.45 ± 0.65 abcd
N at 200 9 3.33 ± 0.33 abc 1.81 ± 0.27 a 3.33 ± 0.33 abc 2.67 ± 0.12 abcde
NPK at 225 9 3.00 ± 0.58 ab 2.35 ± 0.23 ab 5.33 ± 0.67 d 3.88 ± 1.01 e
P at 200 9 5.33 ± 0.58 d 3.23 ± 0.35 bcd 3.00 ± 0.25 ab 2.06 ± 0.25 abc
CK 12 4.00 ± 1.00 bcd 2.58 ± 0.33 ab 2.00 ± 0.25 a 2.62 ± 0.42 abcde
K at 262.5 12 4.00 ± 0.58 bcd 2.69 ± 0.20 abc 3.00 ± 1.00 ab 2.80 ± 0.44 abcde
N at 250 12 4.67 ± 1.20 cd 2.62 ± 0.06 abc 3.67 ± 0.33 bc 3.00 ± 0.43 bcde
NPK at 262.5 12 5.00 ± 1.53 cd 5.15 ± 1.41 e 3.67 ± 0.33 bc 3.41 ± 0.20 cde
P at 250 12 5.33 ± 0.88 d 2.96 ± 0.12 abc 3.33 ± 0.67 abc 3.11 ± 0.19 cde
Values represent the average of three replicates ± standard error of the mean (SEM). Treatment means followed
by different letters in a column are significantly different at p ≤ 0.05 based on the LSD test.
In both experiments, similar vine girths were obtained compared to the control (CK)
plants at the different stages of growth. This indicated that plant age had little influence
on the vine girth. In contrast, Amissah et al. [17] found a significant effect on plant girth
between 9- and 12-month-old C. sanguinolenta.
Furthermore, fertilizer NPK at 225 kg/ha and N at 200 kg/ha produced significantly
higher dry root biomass in the field experiment (22.68 and 22.40 g, respectively) (Table 2).
Although the maximum root biomass yield was obtained in 6-month-old plants in the
pot experiment, the results suggested that fertilizer treatments supplying N (N or NPK)
improved the dry root biomass. Meanwhile, the aboveground biomass was increased by
NPK at 265.2 kg/ha in the field experiment (111.68 g) and NPK at 187.5 kg/ha in the pot
experiment (41.47 g).
Overall, the growth response of C. sanguinolenta to K fertilizer treatments was low but
better than the unfertilized plots. Consequently, the enhanced growth observed in plants
treated with combined NPK fertilizer may be due to a better nutrient balance (i.e., N and
P). Both N and P are important nutrients that significantly affect plant cellular processes
and metabolism. Optimal N supplementation influences plant growth morphology by
increasing cytokinin production for cell elongation and the multiplication of meristematic
cells [29]. Additionally, N increases plant photosynthetic activity and biomass partitioning
between roots and shoots [30]. The study showed that the root biomass of C. sanguinolenta,
the economic plant component, increased when NPK at 225 kg/ha or N at 200 kg/ha
was applied. Such improved production of biomass achieved with fertilizers supplying N
confirms previous reports in Anthriscus cerefolium [31], Cymbopogon martini [27], and Tagetes
minuta [32].
Meanwhile, P addition is needed for cell division and plant metabolism to ensure
maximum plant production [33,34]. The application of P at 250 kg/ha in the present study
increased aboveground biomass (109.47 g), yet still, its effect was statistically similar to that
Plants 2022, 11, 122 4 of 16
obtained by NPK at 262.5 kg/ha (111.68 g) and N at 250 kg/ha (95.10 g) in 12-month-old
plants in the field.
The growth response of C. sanguinolenta was also influenced by the experimental
conditions. For instance, NPK at 262.5 kg/ha recorded the highest aboveground plant
biomass in the field, whereas it was low for the same treatment in the pot experiment. These
differences may be due to the climatic and growth conditions at the experimental sites: the
high rainfall at Bonsu (2.9–140.2 mm) relative to the low rainfall at Legon (14.7–53.6 mm);
the location of Bonsu in the forest zone of Ghana, which is the preferred habitat for
C. sanguinolenta cultivation [35]; limited soil and space in pots for better plant growth.
Table 2. Effects of inorganic fertilizers on biomass yield of C. sanguinolenta at different plant growth
periods.
Treatment Field Experiment Pot Experiment
Plant Age Dry Root Aboveground Dry Shoot/Root Dry Root Aboveground Dry Shoot/Root
Fertilizer (kg/ha)
(in MAT) Biomass (g) Biomass (g) Ratio Biomass (g) Biomass (g) Ratio
CK 3 6.63 ± 0.88 ab 16.10 ± 0.50 a 2.53 ± 0.39 abcd 3.33 ± 0.55 a 4.90 ± 1.15 a 1.44 ± 0.11 ab
K at 150 3 10.87 ± 0.41 bcde 21.37 ± 0.91 ab 1.98 ± 0.14 ab 6.54 ± 1.43 abc 9.73 ± 0.88 ab 1.68 ± 0.50 abc
N at 100 3 13.53 ± 0.54 def 22.80 ± 1.02 abc 1.70 ± 0.14 a 11.18 ± 1.85 defg 15.57 ± 0.33 cd 1.46 ± 0.19 ab
NPK at 150 3 18.97 ± 0.38 ghi 29.57 ± 1.45 cd 1.56 ± 0.07 a 10.94 ± 1.80 defg 25.03 ± 1.15 ghi 2.48 ± 0.59 cde
P at 100 3 6.14 ± 0.29 a 21.00 ± 0.40 ab 3.43 ± 0.12 bcd 6.73 ± 0.33 abc 12.07 ± 1.20 bc 1.81 ± 0.22 abcd
CK 6 10.60 ± 1.01 bcd 27.73 ± 0.60 bcd 2.66 ± 0.23 abcd 6.07 ± 1.98 ab 9.53 ± 1.72 ab 1.71 ± 0.21 abc
K at 187.5 6 12.73 ± 0.19 cdef 35.00 ± 1.25 def 2.75 ± 0.14 abcd 13.37 ± 1.85 fg 15.8 ± 0.50 cd 1.18 ± 0.11 a
N at 150 6 16.27 ± 0.29 fgh 40.97 ± 0.87 ef 2.52 ± 0.01 abcd 13.73 ± 0.24 fg 25.47 ± 0.39 ghi 1.91 ± 0.06 abcd
NPK at 187.5 6 20.67 ± 0.47 i 44.00 ± 0.87 f 2.13 ± 0.01 abc 14.40 ± 1.73 g 41.47 ± 0.94 k 2.95 ± 0.29 e
P at 150 6 12.27 ± 0.70 cdef 31.63 ± 0.95 de 2.60 ± 0.18 abcd 11.40 ± 0.62 defg 16.57 ± 0.45 cde 1.46 ± 0.04 ab
CK 9 8.56 ± 1.66 abc 44.75 ± 4.39 fg 5.43 ± 0.52 f 6.23 ± 0.50 ab 15.85 ± 1.18 cd 2.55 ± 0.10 cde
K at 225 9 11.50 ± 1.80 cde 61.11 ± 8.62 h 5.44 ± 0.47 f 8.39 ± 1.18 bcde 19.32 ± 2.38 def 2.43 ± 0.13 cde
N at 200 9 22.40 ± 2.50 i 75.88 ± 4.66 i 3.45 ± 0.61 bcd 10.22 ± 1.39 cdef 23.77 ± 2.61 fgh 2.42 ± 0.08 cde
NPK at 225 9 22.68 ± 2.88 i 77.67 ± 5.79 i 3.52 ± 0.33 cd 12.17 ± 1.20 efg 25.80 ± 1.18 ghi 2.14 ± 0.45 bcde
P at 200 9 16.07 ± 1.91 fgh 60.19 ± 1.45 h 3.89 ± 0.81 de 8.91 ± 0.97 bcde 21.74 ± 1.59 fg 2.46 ± 0.58 cde
CK 12 10.45 ± 1.17 abcd 56.00 ± 0.40 gh 5.57 ± 0.92 f 8.19 ± 0.98 bcd 21.55 ± 2.23 efg 2.66 ± 0.23 de
K at 262.5 12 12.12 ± 1.63 cdef 74.47 ± 8.11 i 6.51 ± 0.29 f 10.76 ± 1.11 defg 23.98 ± 2.82 fghi 2.22 ± 0.35 bcde
N at 250 12 14.98 ± 1.84 efg 95.10 ± 5.23 j 6.52 ± 0.67 f 12.02 ± 1.30 defg 28.38 ± 3.33 hij 2.46 ± 0.46 cde
NPK at 262.5 12 21.01 ± 2.77 i 111.68 ± 12.24 j 5.36 ± 0.73 ef 13.48 ± 1.88 fg 31.27 ± 2.12 j 2.41 ± 0.52 cde
P at 250 12 19.59 ± 1.44 hi 109.47 ± 7.10 j 5.68 ± 1.38 f 11.12 ± 1.68 defg 29.10 ± 2.95 ij 2.72 ± 0.06 de
Values represent the average of three replicates ± SEM. Treatment means followed by different letters in a column
are significantly different at p ≤ 0.05 based on the LSD test.
2.2. Effect of Inorganic Fertilizers on Cryptolepine Content
The results show that fertilizer application significantly affected cryptolepine content
(mg) in 100 mg of dry root sample compared to unfertilized plants (Figure 1). In the
field experiment, the maximum cryptolepine content (3.14 mg) was achieved with the
application of N at 200 kg/ha, whereas the least (0.57 mg) was obtained in the unfertilized
plants. In the pot experiment, the maximum cryptolepine content was obtained using P
at 200 kg/ha (2.30 mg) or N at 200 kg/ha (2.04 mg). The overall cryptolepine content in
plants grown in the field was on average 61.4% higher than those grown in pots.
The observed positive effects of nitrogen application on the biosynthesis of the target
secondary metabolite in C. sanguinolenta confirms previous reports of similar effects in
Datura innoxia [25], Catharanthus roseus L. [24], Ocimum basilicum L. [36], Foeniculum
vulgare Mill. [37], medicinal Pumpkin [38], Origanum vulgare L. [39], Cymbopogon martinii
Roxb. [27] and Rosmarinus officinalis L. [40]. One possible explanation for this outcome is
that N is a major component in cryptolepine, an indole alkaloid containing nitrogen on its
heterocyclic system, and a precursor for proteins and enzymes that play a key role in alka-
loid biosynthesis and accumulation in plant roots [41]. Another reason may be attributed
to the high rate of N supplied (200 kg/ha) leading to higher photosynthesis, division, and
elongation of new cells that are alkaloid secretory ducts and storage glands [42].
Although N (at 200 kg/ha) was the most promising, the results showed that P applica-
tion enhanced cryptolepine production relative to the unfertilized plants in the 9-month
growth period. Ramezani et al. [43] found that the supply of P greatly increased the
PPllaannttss 22002212,, 1101,, x12 F2OR PEER REVIEW 5 5ooff 1176 
 
Iens stehnet ipaol to eilxcpoenritmenetnot,f tOhe.  bmasaixliicmumum. S cimryiplatorleefpfiencets cwonetreenot bwsearsv oebdtaininMeda tursicinargi aPr eactu 2ti0t0a 
kbgy/hJeas (h2n.3i0e tmalg.)[ 4o4r] .NI ant c2o0n0t rkags/th, at h(e2.0e4ss mengt)i.a lTohiel ocvoenrtaelnl tcirnypLtaovleanpdinuela coanngteunstti fionl ipa lManitlsl. 
gprroowdunc iend thuen dfieerldh ywdarso opno naivcecroangde i6ti1o.n4%s r hemigahienre tdhaunn athffoescete gdrobwy nP isnu pppoltys. [45].
 
Figure 1. Cryptolepine conteFnigt uinr ero1o. tCs royfp Ct.o sleapnignueincoonletnetnat iinn rreosoptsonofseC t.os afenrgtuiliinzoelre ntrteaaitnmreensptso wnsiethtionf feorutirli gzreorwtrteha tpmereinotdssw. ithin
The growth periods/plant agfoeus rarger oinw mthopnethriso adfst.eTr htreangsropwlatnhtipnegr (iModAs/Tp),l aanntda CgKes, aKr,e Nin, NmPoKn,t hasndaf Pte rdetrnaontsep tlhaen ttirnegat(mMeAntTs) , and
applied: Control, PotassiumC, KN,iKtr,oNge,nN, PcKom, abnidnePd dNenPoKt,e atnhde  tPrehaotsmpehnotrsuasp, prelisepde:cCtivoneltyro. lB, aProst aresspiruemse,nNt itthroe gmene,acno omfb tihnreede NPK,
replicates, and error bars araen tdheP ShEosMp.h Boarurss ,hraevspinegct tivheel ys.aBmaer scoreloprr efsoelnlotwtheedm beya dnifoffetrhernete lerettpelrisc aatrees ,saignndifeircraonrtlbya rdsifafreerethnet SEM.
at p ≤ 0.05 based on the LSDB taersst.h aving the same color followed by different letters are significantly different at p ≤ 0.05 based
on theThLSeD obtessetr.ved positive effects of nitrogen application on the biosynthesis of the target 
secondary metabolite in C. sanguinolenta confirms previous reports of similar effects in 
D2.a3t.uErfafe icnt nofoGxirao w[2t5h]P, CeraiotdhsaorannCtrhyupst orloespeinues CLo. n[2te4n],t Ocimum basilicum L. [36], Foeniculum 
vulgaAres iMdeilflr. o[m37]f,e rmtielidziecrinaaplp Pliucamtipokni,np [la3n8]t,a Ogeri(ginanMumAT v)ualtghaarrev Le.s t[3s9ig],n Cifiycmanbtolypoingfloune mncaerd-
ttihneiic Rryopxtbo.l [e2p7i]n aencdo nRtoesnmt ianritnhuesr oofoftisci(nTaalbisle L3. [a4n0d]. FOignue rpeo1s)s.ibInlet ehxepfilaenldateioxnp efroirm thenist ,otuhte-
ccorympet oisl etphiant eNc oisn ate mntaijonrc rceoamsepdonweintht ipnl acrnytpatgoele, pfrionme, a0n.8 i8nmdoglei nal3k-amlooidnt cho-noltdainpilnagn tnsittoroa-
gpeena kono fit2s. 6h3etmergociync9li-cm syosnttehm-o, ladnpdl aan ptrse. cCurrsyoprt ofolerp pinroetecionnst eanntdd eenczryemaseesd thslaigt hptllayy ian ktehye 
r1o2l-em ino natlhk-aololdidp blaionstysnatnhdeswisa asnldik aeclycutmheurleastiuolnt  oinf  spelnanest creonoctes [a4t1t]h. iAs ngorothwetrh repaesroiond mwahyi bche 
artetdriubcuetdedth teom thetea bhoiglihc aracttiev iotfy Nof sthueppplliaendt o(2rg00a nksg. /Shima) illaeradtrienngd tsow heirgehoebr sperhvoetdosiynntthheepsiost, 
deixvpiesiroimn,e anntd.  Tehloenfignatdioinng osfo nnewpl acneltlsa gtheact oarrreo ablokraaltoeidA smecisrseatohryet daul.ct[s1 7a]n, dw shtoorcaognes gidlaenredds 
[94-2m].o nth-old C. sanguinolenta appropriate for harvesting.
Although N (at 200 kg/ha) was the most promising, the results showed that P appli-
cation enhanced cryptolepine production relative to the unfertilized plants in the 9-month 
growth period. Ramezani et al. [43] found that the supply of P greatly increased the es-
sential oil content of O. basilicum. Similar effects were observed in Matricaria recutita by 
 
Plants 2022, 11, 122 6 of 16
Table 3. Mean cryptolepine content and yield at different harvest periods.
Field Experiment Pot Experiment
Plant Age at
Harvest (in MAT) Cryptolepine Content (mg) Cryptolepine Yield Cryptolepine Content (mg) Cryptolepine Yield
in 100 mg of Dry Roots (mg/plant) in 100 mg of Dry Roots (mg/plant)
3 0.88 ± 0.14 a 106.70 ± 33.86 a 0.63 ± 0.07 a 49.94 ± 11.98 a
6 1.26 ± 0.08 b 186.00 ± 29.33 a 0.78 ± 0.12 a 91.50 ± 18.56 b
9 2.63 ± 0.27 c 431.06 ± 91.36 b 1.88 ± 0.17 c 177.10 ± 30.95 c
12 2.61 ± 0.28 c 413.90 ± 81.98 b 1.28 ± 0.16 b 146.60 ± 31.44 c
mean 1.84 ± 0.19 284.39 ± 42.91 1.14 ± 0.12 116.26 ± 14.42
Values represent the average of five replicates ± SEM. Treatment means followed by different letters in a column
are significantly different at p ≤ 0.05 based on the LSD test.
2.4. Effect of Treatment Combination (Fertilizers and Growth Periods) on Cryptolepine Yield
Further analysis between the treatments revealed that cryptolepine yields (mg/plant)
in both experiments were significantly enhanced by N or NPK treatments at each growth
period (Figure 2). The average cryptolepine yield in the field was 284.4 mg and ≈2.5-fold
Plants 2021, 10, x FOR PEER REVIEW 
 higher than pot-grown plants (116.3 mg) (Table 3). This result highlighted the po
7te onft i1a7l 
economic gain for producing the herb under field conditions.
 
Figure 2. Cryptolepine yieldF iing urroeo2ts. oCfr Cyp. tsoalnegpuinineoyleienltda iinn rroeosptsoonfseC .tosa fnegrtuiilnizoelre nttraeaintmreesnptso nwsiethtionf feorutirli gzerorwtrteha tpmereinotdssw. ithin
The growth periods/plant agfoesu rarger oinw mthopnethriso adfste. rT threangsrpolwanthtinpge r(iModAs/Tp), laanndt  aCgKes, Kar,e Nin, NmPoKn,t hansda fPte dretrnaontesp thlaen ttrineagtm(MeAntTs) , and
applied: Control, PotassiumC, KN,itKro, Nge,nN, PcoKm, abnindePd dNePnKot,e atnhde tPrheaotsmphenotrsuas,p rpelsiepde:ctCivoenltyr.o Bl,aPros traespsiruemse,nNt itthreo gmenea, nco omf bthinreede NPK,
replicates, and error bars are the SEM. Bars having the same color followed by different letters are significantly different 
at p ≤ 0.05 based on the LSDa tnedst.P hosphorus, respectively. Bars represent the mean of three replicates, and error bars are the SEM.
Bars having the same color followed by different letters are significantly different at p ≤ 0.05 based
on thIenLteSrDestetisnt.gly, the increase in cryptolepine yield paralleled the increase in root bio-
mass in the 9-month-old plants. This was evident in both trials’ significant positive corre-
lation between plant biomass yield and cryptolepine yield (Table 4). Thus, substances that 
improve root growth and biomass are presumed to increase cryptolepine yield. The eco-
nomic yield of C. sanguinolenta integrates the production of root biomass and cryptole-
pine synthesis, and both were increased by N fertilization in the present study. As a result, 
the increase in cryptolepine yield with NPK fertilization could be due to the availability 
of N in this fertilizer since the root biomass yield for K or P applied alone was relatively 
low. These findings are consistent with those recorded in Basil, Palmarosa, and Fennel 
[26–28]. They found significant improvement in biomass production and expansion of the 
leaf area, resulting in greater essential oil yield. 
Table 4. Correlation between plant biomass, vine number, girth, cryptolepine content and yield in C. sanguinolenta. 
 Root  Shoot  Vine  Vine Girth Cryptolepine Cryptolepine 
Biomass Biomass Number Content Yield 
Root biomass  0.78 ** 0.57 ** 0.83 ** 0.16 0.53 * 
Shoot biomass 0.61 **  0.46* 0.71 ** 0.39 0.65 ** 
Vine number 0.53 * 0.57 **  0.65 ** 0.01 0.19 
Vine girth 0.25 0.05 0.49 *  0.25 0.54 * 
Cryptolepine content 0.50 * 0.88 ** 0.50 * −0.20  0.89 ** 
Cryptolepine yield 0.82 ** 0.88 ** 0.50 * −0.03 0.87 **  
 
Plants 2022, 11, 122 7 of 16
Interestingly, the increase in cryptolepine yield paralleled the increase in root biomass
in the 9-month-old plants. This was evident in both trials’ significant positive correlation
between plant biomass yield and cryptolepine yield (Table 4). Thus, substances that im-
prove root growth and biomass are presumed to increase cryptolepine yield. The economic
yield of C. sanguinolenta integrates the production of root biomass and cryptolepine syn-
thesis, and both were increased by N fertilization in the present study. As a result, the
increase in cryptolepine yield with NPK fertilization could be due to the availability of N
in this fertilizer since the root biomass yield for K or P applied alone was relatively low.
These findings are consistent with those recorded in Basil, Palmarosa, and Fennel [26–28].
They found significant improvement in biomass production and expansion of the leaf area,
resulting in greater essential oil yield.
Table 4. Correlation between plant biomass, vine number, girth, cryptolepine content and yield in C.
sanguinolenta.
Root Shoot Vine Vine Girth Cryptolepine CryptolepineBiomass Biomass Number Content Yield
Root biomass 0.78 ** 0.57 ** 0.83 ** 0.16 0.53 *
Shoot biomass 0.61 ** 0.46 * 0.71 ** 0.39 0.65 **
Vine number 0.53 * 0.57 ** 0.65 ** 0.01 0.19
Vine girth 0.25 0.05 0.49 * 0.25 0.54 *
Cryptolepine content 0.50 * 0.88 ** 0.50 * −0.20 0.89 **
Cryptolepine yield 0.82 ** 0.88 ** 0.50 * −0.03 0.87 **
* and ** show a significant correlation at alpha levels 5 and 1%, respectively. The non-bold-faced and bold-faced
coefficients show correlations in the field and pot experiments, respectively.
2.5. Comparison of Variations in Cryptolepine Yield Due to Fertilizer Application and
Growth Period
In both experiments, the nested ANOVA method showed significant variations in
cryptolepine yield due to the growth periods and fertilizers applied (Table 5). However,
a large variation in the broad sense signifies the factor that is of utmost importance for
optimum resource allocation. The variance component study revealed that little variation
in cryptolepine yield occurred between fertilizer treatments (33.29% in the field experiment
and 29.18% in the pot experiment), as well as between treatment replicates. Much of the
observed variations occurred between the growth periods (55.67% in the field and 50.63%
in the pot experiments), indicating that growth periods determined the cryptolepine
yield even more than fertilizer application. Therefore, plant age must be considered
first (9-month cultivation), followed by N fertilizer application for optimum productivity
and cryptolepine yield. Our results are consistent with previous reports where several
factors, including plant age and nutrition, influenced the yield of alkaloids in different
plants [24,46–48].
Table 5. An estimate of variance due to growth periods and fertilizer treatments on cryptolepine
yield in the field and pot experiments.
Source of Variation Degrees of Sum of MeanFreedom Squares Squares F-Value p-Value
Variance
Component % of Total
Field Experiment
Growth period (GP) 3 1167.58 389.19 8.53 0.001 22.90 55.67
Block 2 3.44 1.72 0.37 0.696 0.00 0.00
Fertilizer (GP) 16 730.07 45.63 9.72 0.000 13.70 33.29
Error 38 178.32 4.69 4.54 11.04
Total 59 2079.41 41.14 100.00
Pot Experiment
Growth period (GP) 3 347.20 115.73 9.26 0.001 6.76 50.63
Fertilizer (GP) 16 200.10 12.50 3.61 0.000 3.90 29.18
Error 40 138.40 3.46 2.70 20.19
Total 59 685.70 13.36 100.00
Variance component was estimated using the nested method of ANOVA: fertilizer treatments were nested within
the growth periods, p-values < 0.05 are significant at 5% alpha level.
Plants 2022, 11, 122 8 of 16
2.6. Effects of Inorganic Fertilizer Application on Soil Fertility
The initial soil analysis showed that the soil used in this study had low total N,
available K, P, and organic matter content, implying low soil fertility (Table 6). This,
therefore, justified the need for amendment with fertilizer to improve the fertility status.
Table 6. Physical and chemical characteristics of soil at 0–20 cm depth before planting.
Avail. P Exchangeable Cations (cmol/kg) Particle Size (%)pH(H2O) (mg/kg) % N % OC K+ Na+ Ca2+ Mg2+ Sand Silt Clay
5.50 9.72 0.09 0.60 0.14 0.70 2.60 2.00 52.90 22.10 25.00
The effect of fertilizer application on the soil macronutrient levels (N, P, and K) in
the field and pot experiments at the end of the harvesting periods were compared. In
the field experiment, the fertilizers applied significantly affected the macronutrient levels
at the end of the harvesting period (Table 7). The highest amount of residual N in the
soil after harvesting was obtained when N fertilizer at 100 kg/ha was applied, while the
corresponding amount of residual K was obtained when K at 150 kg/ha was applied.
Generally, the levels of post-harvest residual N and K decreased after the 3-month growth
period. However, in the case of P, application of NPK at 225 kg/ha significantly increased
the total P in both the field and pot experiments, compared with the application of P
fertilizer alone.
Table 7. Effects of inorganic fertilizer application on soil macronutrients (N, P, and K) levels at the
end of the different harvesting periods.
Treatment Field Experiment Pot Experiment
Fertilizer Plant Age
(kg/ha) (months) Total N (%) P (mg/kg) K (cmol/kg) Total N (%) P (mg/kg) K (cmol/kg)
CK 3 0.84 ± 0.37 b 9.62 ± 0.58 ab 1.10 ± 0.58 cd 1.40 ± 0.20 b 17.54 ± 0.58 a 1.50 ± 0.33 b
K at 150 3 1.42 ± 0.33 c 29.77 ± 0.66 f 3.02 ± 0.33 f 1.64 ± 0.39 b 16.17 ± 0.58 a 1.71 ± 0.33 b
N at 100 3 1.93 ± 0.42 d 16.29 ± 0.61 bcd 1.09 ± 0.58 bcd 1.68 ± 0.31 b 16.36 ± 0.58 a 1.46 ± 0.33 b
NPK at 150 3 1.15 ± 0.58 cd 72.50 ± 0.58 i 2.12 ± 0.37 e 1.43 ± 0.33 b 65.28 ± 0.58 g 1.69 ± 0.33 b
P at 100 3 1.50 ± 0.30 cd 39.24 ± 0.58 g 1.48 ± 0.30 d 1.34 ± 0.33 b 42.02 ± 0.58 cd 1.46 ± 0.33 b
CK 6 0.07 ± 0.00 a 29.00 ± 0.73 ef 0.10 ± 0.01 a 0.07 ± 0.03 a 14.43 ± 0.38 a 0.13 ± 0.00 a
K at 187.5 6 0.06 ± 0.00 a 19.78 ± 0.85 cde 0.50 ± 0.02 ab 0.07 ± 0.01 a 14.49 ± 1.31 a 0.31 ± 0.01 a
N at 150 6 0.07 ± 0.00 a 25.11 ± 0.83 def 0.09 ± 0.01 a 0.07 ± 0.00 a 12.20 ± 0.23 a 0.11 ± 0.10 a
NPK at 187.5 6 0.09 ± 0.00 a 54.51 ± 1.06 h 0.33 ± 0.02 a 0.09 ± 0.00 a 56.03 ± 2.63 ef 0.19 ± 0.01 a
P at 150 6 0.06 ± 0.00 a 75.13 ± 4.07 i 0.10 ± 0.01 a 0.06 ± 0.03 a 34.90 ± 1.07 bc 0.13 ± 0.00 a
CK 9 0.16 ± 0.01 a 8.03 ± 0.64 ab 0.09 ± 0.03 a 0.10 ± 0.01 a 17.87 ± 1.34 a 0.15 ± 0.01 a
K at 225 9 0.13 ± 0.01 a 6.72 ± 1.93 a 0.55 ± 0.05 abc 0.09 ± 0.01 a 48.80 ± 5.21 de 0.20 ± 0.03 a
N at 200 9 0.12 ± 0.02 a 7.78 ± 1.49 ab 0.09 ± 0.01 a 0.10 ± 0.03 a 54.31 ± 5.71 ef 0.13 ± 0.01 a
NPK at 225 9 0.15 ± 0.01 a 93.35 ± 1.70 j 0.44 ± 0.05 a 0.09 ± 0.00 a 76.68 ± 3.02 h 0.20 ± 0.01 a
P at 200 9 0.13 ± 0.02 a 70.40 ± 12.93 i 0.09 ± 0.01 a 0.10 ± 0.01 a 57.20 ± 5.09 f 0.14 ± 0.01 a
CK 12 0.12 ± 0.01 a 13.10 ± 1.31 abc 0.05 ± 0.01 a 0.15 ± 0.01 a 11.97 ± 1.06 a 0.05 ± 0.03 a
K at 262.5 12 0.12 ± 0.01 a 16.36 ± 1.81 bcd 0.35 ± 0.07 a 0.15 ± 0.00 a 10.55 ± 0.91 a 0.15 ± 0.02 a
N at 250 12 0.12 ± 0.01 a 9.32 ± 0.85 ab 0.06 ± 0.00 a 0.20 ± 0.01 a 14.31 ± 1.39 a 0.07 ± 0.01 a
NPK at 262.5 12 0.09 ± 0.01 a 43.20 ± 1.12 g 0.12 ± 0.01 a 0.16 ± 0.01 a 51.53 ± 3.77 ef 0.08 ± 0.03 a
P at 250 12 0.10 ± 0.00 a 20.12 ± 1.72 cde 0.07 ± 0.01 a 0.16 ± 0.03 a 31.15 ± 3.17 b 0.06 ± 0.01 a
Values represent the average of three replicates ± SEM. Treatment means followed by different letters in a column
are significantly different at p ≤ 0.05 based on the LSD test.
Although the soil nutrients were estimated before planting and after absorption by
crops, the influence of fertilizer treatment regimens on soil nutrients (N, P, and K) at the end
of the harvesting period may be due to the fertilizers ability to supply soil nutrients readily
for use during plant growth and development. These findings agree with studies in which
the soil fertility status was improved remarkably by using inorganic fertilizers [22,29,32].
Yousaf et al. [49] noted that P supply and uptake could be optimized when balanced
mineral NPK is adopted for soil modification in rapeseed oil cultivation. In the present
study, the increase in soil P fraction following the application of NPK rather than P could
be due to the synergistic effect of N or K on P mineralization [50]. In addition, the presence
of metal oxides (Al and Fe) could influence the adsorption and precipitation of P [51].
Plants 2022, 11, 122 9 of 16
In the pot experiment, fertilizer treatments within the same growth period had no
significant effect on the levels of macronutrients. This may be due to the extensive use of
these supplied nutrients by plants as they mature rapidly in the limited space in the pots.
2.7. Effect of Growth Periods on the Bioactivity of C. sanguinolenta Extracts
The bioactivity of cryptolepine from C. sanguinolenta plants at different growth periods
was tested in vitro against P. falciparum strain Dd2. Table 8 shows the concentrations of
extracts that can inhibit 50% of plasmodium infected RBCs. The antiplasmodial activ-
ity of root extracts from 3-, 6-, and 9-month-old plants in pots was moderately potent
(IC50 = 6.30–9.23 µg/mL). However, root extracts from 12-month-old plants showed the
highest potency (2.56 µg/mL) with about two to three-fold stronger antiplasmodial activity.
On the other hand, 9-month-old plants showed the highest potency (4.65 µg/mL) in the
field experiment.
Table 8. The effects of plant growth period on the antiplasmodial and anti-cancer activity of C.
sanguinolenta extracts.
Antiplasmodial Activity Antiplasmodial Selectivity Index (SI) Anti-Cancer Activity(Cell Viability)
Growth Period
(in MAT) IC50 Values for IC50 Values forPot Expt. Field Expt. SI for Pot Expt. SI for Field Expt.
CC50 Values for CC50 Values for
(*CC /IC ) (*CC /IC ) Pot Expt. Field Expt.(µg/mL) (µg/mL) 50 50 50 50 (µg/mL) (µg/mL)
3 9.23 ± 0.05 8.12 ± 0.07 1.08 1.23 6.83 ± 1.03 12.25 ± 1.41
6 7.37 ± 0.06 6.44 ± 0.56 1.36 1.55 30.91 ± 1.95 8.81 ± 0.76
9 6.30 ± 0.30 4.65 ± 0.02 1.59 2.15 11.7 ± 2.52 8.68 ± 0.46
12 2.56 ± 0.04 5.06 ± 1.28 3.91 1.98 – 62.53 ± 6.68
Control 3.01 ± 0.03 x 3.01 ± 0.03 x 0.05 0.05 8.56 ± 1.67 y 8.56 ± 1.67 y
* Because the 50% cytotoxic concentrations (CC50) for all extracts were outside the range of extract concentrations
tested, the highest sample concentration (10 µg/mL) was considered as the CC50 for calculating the selectivity
index (SI). x and y represent values from controls: chloroquine and curcumin, respectively. Missing data (–).
Interestingly, the results from both experiments showed increased antiplasmodial
activity as the plants aged. This follows the increasing cryptolepine content observed with
plant age in the previous experiment (Table 3), indicating the relevance of plant age on the
effectiveness of C. sanguinolenta decoctions for malaria treatment.
An independent-samples t-test was performed to compare the antiplasmodial activity
of extracts obtained in both experiments: field versus pot. There was no significant effect for
production medium, t (6) = 0.18, p = 0.859, despite the slightly higher antiplasmodial activ-
ity of plants in the field (average IC50 = 6.07 ± 0.78) than potted plants (IC50 = 6.37 ± 1.41).
The results suggest that C. sanguinolenta can be produced either in the field or in pots
without losing antimalarial activity. However, the field environment must be consid-
ered for production because it represents the plant’s natural habitat, promotes biomass
accumulation, and increases cryptolepine yield based on the present findings.
Furthermore, the concentrations needed to reduce RBC survival by 50% (CC50) for
all the extracts tested were found to be >10 µg/mL, while that for chloroquine was
0.15 ± 0.00 µg/mL and more toxic to RBCs. All the extract concentrations tested were
non-cytotoxic to RBCs and thus exhibited high drug selectivity (Table 8). The high selective
index for extracts collected from 9-month-old plants in the field (2.15) and 12-month-old
plants in pots (3.91) confirmed that plants cultivated under these two growth conditions
were the most effective against P. falciparum Dd2.
Aside from antiplasmodial activity, the root extracts were cytotoxic to Jurkat leukaemia
cell lines in vitro, with CC50 values <30.91 µg/mL for the field and <62.53 µg/mL for the
pot samples. Although the impact of plant age on anti-cancer activity was not clear, the
findings indicated the possible use of cryptolepine as an agent for cancer management and
further strengthened previous reports on other human cancer cells [52–54].
Plants 2022, 11, 122 10 of 16
3. Materials and Methods
3.1. Study Sites
Two trials were carried out concurrently from April 2013 to July 2014 to investigate
the effects of inorganic fertilizers on C. sanguinolenta. The field experiment was set up at
the Centre for Scientific and Industrial Research–Plant Genetic Resource Research Institute
(CSIR-PGRI) farm located in Bunso (N 5◦46′, W 1◦1′, 204 m above sea level), a tropical
rainforest zone with about 1565 mm rainfall and 25.9 ◦C temperature annually. The
pot experiment was set up at the University of Ghana Research Farm located in Legon
(N 5◦39′, W 0◦11′, 37 m above sea level), a coastal savannah zone with about 809 mm
rainfall and 26.6 ◦C temperature annually. Weather conditions during the study period at
Bunso were as follows: rainfall (2.9–140.2 mm), temperature (20.6–34.9 ◦C), and relative
humidity (75.5–87.5%). The conditions at Legon were: rainfall (14.7–53.6 mm), temperature
(23.4–32.8 ◦C), and humidity (73.1–86.0%). The field was initially prepared by ploughing
and harrowing before raising mounds of 25 to 35 cm high and spaced at 80 × 80 cm.
The soil used in the study was typically a Haplic Lixosol belonging to the Kokofu
soil series of Ghana [55], according to the FAO-UNESCO classification [56]. The soil
was analyzed for pH (1:1) and exchangeable cations (Na+H2O , Ca+, and Mg2+) using the
ammonium acetate extraction method as outlined by Page et al. [57]. The particle-size
distribution was determined according to the method described by Bouyoucos [58]; soil
organic carbon (OC) according to Black [59]; total N content of the soil by the Kjeldahl
method [60]. The available P was determined using the Lamba 45 spectrophotometer
(Perkin Elmer, Inc. USA) after extraction in sodium hydrogen carbonate [61]. The K content
was determined using a flame photometer (Jenway PFP7, Cole-Parmer Ltd. Staffordshire,
UK) after digestion with ammonium acetate [62].
For the pot experiment, topsoil collected from the field research site at Bunso was
thoroughly mixed and oven-sterilized at 72 ◦C for 3 days. Next, 9.6 kg of soil was filled
into 10 L plastic pots and spaced at 80 × 80 cm. These were watered and allowed to drain
overnight before transplanting.
3.2. Transplanting of Seedlings
C. sanguinolenta seeds (accession #201 KA), obtained from the CSIR-PGRRI, were sown
directly into nursery containers filled with topsoil and watered. Twelve days after sowing,
seedlings emerged and were thinned three weeks later. Healthy 12-week-old uniform
seedlings were transplanted onto the mounds (600 seedlings, one seedling per mound) and
pots (420 seedlings, one seedling per pot) in July 2013. The transplanted seedlings were
irrigated regularly for two weeks. Afterward, irrigation was performed as required based
on rainfall.
3.3. Field Experiment
Four inorganic fertilizers, namely Urea (supplying 46% N), Triple Super Phosphate
(46% P2O), Muriate of Potash (60% K2O), combined NPK (15-15-15%), and a control
(without fertilizer) were studied; their nomenclatures are N, P, K, NPK, and CK, respectively.
Fertilizers were nested within four plant growth periods (3, 6, 9, and 12 months after
transplanting (MAT)), giving 20 experimental treatments in total. Treatments were assigned
to plots based on a randomized complete block design with three replicates. Each replicate
contained ten plants. The fertilizer application rate was divided equally and added monthly
during the growth period using the band placement method at a depth of 5 cm and 10 cm
away from the plant (Table 9).
Plants 2022, 11, 122 11 of 16
Table 9. Treatments and fertilizer rates used in the field and pot experiment.
Fertilizer Rates (kg/ha) Used in Each Growth Period (Months
Fertilizer after Transplanting)
3 Months 2* 6 Months 4 9 Months 6 12 Months 8
Urea (N) 100.0 150.0 200.0 250.0
Triple Super Phosphate (P) 100.0 150.0 200.0 250.0
Muriate of Potash (K) 150.0 187.5 225.0 262.5
NPK 150.0 187.5 225.0 262.5
Control (CK) 0.0 0.0 0.0 0.0
* Number of times fertilizer (split dose) was combined within each growth period at monthly intervals.
3.4. Pot Experiment
The field experiment was repeated using potted plants with minor modifications to
the setup: treatments were completely randomized in all units; each replicate contained
five plants; fertilizers were spread widely on the soil surface and stirred into the soil using
a hand fork. During the growing season, both the field plots and the pots were regularly
cleared of weeds.
3.5. Plant Growth Measurements, Crop Harvest, and Biomass Analysis
Eight record plants from a treated plot were harvested once plant maturation met a
particular growth period (Table 9). All plants in the pot experiment were used as record
plants. The number of vines that developed from the main stem was counted, and the girth
of two to three vines was measured with a veneer caliper and recorded. Harvesting was
performed manually by digging up the plant along with its roots. Subsequently, the root
system was detached from the shoot, and the dry biomass recorded after oven drying the
samples at 70 ◦C to a constant weight.
3.6. Soil Analysis
Soil analysis was performed to determine the macronutrient levels in the soil. Soil
samples were taken at harvest from the top of five mounds and five pots to a depth
(≈15–25 cm), using a soil auger (10 cm barrel and 5 cm diameter). The composite sample
was thoroughly mixed and then sieved through a 0.25 mm mesh. The total N, P, and
K content in the soil sample was determined as previously described (Section 3.1). The
experiment was replicated three times (n = 3), using a fresh sample each time.
3.7. Estimation of Cryptolepine by High-Performance Liquid Chromatography (HPLC)
Of the eight record plants used to assess plant biomass, three roots were set aside and
air-dried under a shade at room temperature (25 ◦C) for 1 month, then ground together
into a fine powder. An amount of 100 mg of the ground sample was soaked in absolute
ethanol (50 mL) at 25 ◦C for 24 h and filtered using a Whatman filter paper. Three replicate
extractions (n = 3) were made for each treatment. The extracts obtained were evaporated
to dryness in a Rotavapor R114 evaporator (Buchi Labortechnik AG, Fawil, Switzerland),
then reconstituted in 20 mL absolute ethanol and stored at 4 ◦C until use.
The cryptolepine content in the extract was analyzed by HPLC at the Department of
Chemistry, University of Ghana, Legon, using the Varian 920-LC Analytical HPLC system
(Varian Inc., Palo Alto, USA) and a Pursuit C18 (5 µm particle size) column (250 × 4.6 mm
id; Varian; Cat. No. 1215–9307). The mobile phase was an HPLC grade methanol/water
(v/v, 90:9) adjusted to a pH of 2.4 using dichloroacetic acid (DCA). The analytical conditions
and methods of the chromatographic assay were performed as described by Amissah
et al. [17]. Cryptolepine was quantified with the help of a standardized curve obtained by
HPLC, using different concentrations of standard cryptolepine (Sigma-Aldrich Corp., St.
Louis, MO, USA) dissolved in absolute ethanol. The results obtained were expressed in mg
Plants 2022, 11, 122 12 of 16
of cryptolepine content/100 mg of dry ground roots. The economic yield was calculated
according to the equation:
Cryptolepine yield (mg plant−1) = Dry root biomass (g plant−1) ×
(1)
Cryptolepine content (mg/100 mg of ground dry roots) × 1000 (mg/g).
3.8. Bioefficacy of Cryptolepine Extracts
The efficacy of the active ingredient (Cryptolepine) in the roots of C. sanguinolenta
plants harvested at different ages was determined against malaria parasite and Jurkat cells
at the Noguchi Memorial Institute for Medical Research, Accra, Ghana.
A chloroquine-resistant strain (Dd2) of Plasmodium falciparum was maintained in
continuous culture by the modified method of Trager and Jensen [63]; RPMI 1640 medium
containing Hepes (N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid), and sodium
bicarbonate (NaHCO3), but without glutamine (Sigma Chemical Co., St. Louis, MO,
USA), was used. The medium was supplemented with 10% normal human serum (NHS),
1 mg/mL of L-Glutamine, 1 mg/mL of D-Glucose (Sigma-Aldrich Corp. St. Louis, MO,
USA), and 80 µg/mL of gentamicin (Gibco BRL Life Technologies, Paisley, Scotland). After
informed consent, volunteers from the blood group O Rh+ donated whole blood for the
study. All other chemicals were of analytical grade and obtained from the companies
mentioned above.
3.8.1. Antiplasmodial Activity of C. sanguinolenta Extracts
The antiplasmodial activity of extracts obtained from unfertilized plants (CK) of each
harvest period (3, 6, 9, and 12 MAT) in the field and pot experiments were analyzed in vitro
using the SYBR® Green assay, with slight modification [64]. First, dilutions of each of the
test samples (0–10 µg/mL) were prepared in RPMI 1640 culture medium (supplemented
with L-glutamine, gentamicin, albumax preparation, and glucose). Subsequently, 100 µL
of P. falciparum-infected RBCs (2.3 × 108 cells/mL) at a parasitemia of 1.5% was added to
each well. Chloroquine was used as a positive control. All plates were transferred into
a humidified candle jar and incubated at 37 ◦C for 72 h with 5% CO2. Next, a 100 µL of
SYBR® Green solution (diluted with lysis buffer) was added to each well and incubated
in the dark at room temperature for 2 h. The SYBR® Green fluorescence was measured
with a Tecan Infinite M200 microplate reader (Tecan Austria GmbH, Grodig, Austria)
with excitation and emission wavelength at 485 and 530 nm, respectively. The extract
concentration (IC50) causing 50% inhibition of P. falciparum growth was obtained from
the drug concentration-response curve and the mean IC50 determined from experiments
performed in triplicate (n = 3).
3.8.2. Cytotoxicity of C. Sanguinolenta Extracts
A modified version of the tetrazolium-based colourimetric assay [65] was used to test
samples for their antiplasmodial activity and toxicity to RBCs. Dilutions of each test sample
(0–10 µg/mL) were prepared in appropriate solvents and placed in separate wells of a
96-well microtitre plate in triplicates. Subsequently, 100 mL of P. falciparum-infected RBCs
(2.3 × 105 cells per ml) at a parasitemia of 1.5% was added to each well. The contributions
of plant medicine, parasite culture medium, infected and uninfected RBCs to the optical
densities were excluded by setting up control experiments for each of these parameters
separately alongside the main experiments. Chloroquine was used as a positive control.
All plates were transferred into a candle jar and incubated at 37 ◦C for 5 days under
low levels of O2 and CO2 after which 2.5 mg/mL MTT (3-(4,5-Dimethylthiazol-2-YI)-2,5-
Diphenyltetrazolium Bromide) reagent was added to each well, and the plates incubated
again for 2 h. A stopping solution (200 µL of acidified isopropanol) was added to each well
to dissolve any formazan formed. The experiment was repeated thrice for each sample
concentration. The plates were then kept at room temperature in the dark for 24 h, and
the optical densities (OD) read at a wavelength of 570 nm using the Tecan Infinite M200
Plants 2022, 11, 122 13 of 16
microplate reader. The percentage protection and survival of RBCs from P. falciparum
destruction were calculated using the modified formula by Ayisi et al. [65]. The values
were plotted against the concentrations of extracts, and the concentration causing 50%
cell survival (cytotoxic concentrations, CC50) was determined. The selective indices of the
extracts were also computed as the ratio of CC50 to IC50 value.
3.8.3. Anti-Cancer Activity of C. sanguinolenta Extracts
The anti-cancer properties of the extracts were also evaluated by assessing their
cytotoxic effects on human Jurkat cell lines using the MTT assay [65]. Jurkat cell lines
were cultured as described by Appiah-Opong et al. [66] and incubated in a humidified
chamber at 37 ◦C and 5% CO2. The cells were seeded into a 96 well plate (1× 104 cells/well).
Subsequently, the cells were treated with varying extract concentrations (0–100 µg/mL).
Curcumin was used as a positive control. After incubating the plate for 72 h, 20 µL of MTT
solution (2.5 mg/mL in PBS) was added to each well and further incubated for 4 h. Aliquots
of acidified isopropanol (200 µL) were added to each well, and the optical densities were
read as described in the previous experiment. The average cytotoxic concentration (CC50)
was determined from experiments performed in triplicate (n = 3).
3.9. Data Analysis
Data collected were checked for normality and homogeneity of variances before
using the ANOVA test. When the results showed statistical significance (p-value ≤ 0.05),
heterogeneous group means were separated with the least significant difference test (LSD).
A variance component analysis was performed to estimate the relative contribution of
growth periods and fertilization to the total variation in cryptolepine yield by nesting the
fertilizer treatments within the growth periods. Additionally, the relationship between
various plant parameters in the study was calculated using the Pearson correlation index
(r). Results were presented as means and their standard errors. All statistical tests were
carried out at a 5% significance level in Genstat V12 statistical package [67].
4. Conclusions
The study showed that mineral fertilization and plant age at harvest positively affect
the cryptolepine yield in C. sanguinolenta. N application (with or without P and K) increases
root biomass yield, cryptolepine content, and cryptolepine yield. The 9-month growth
period was confirmed as optimal for harvesting the herb and determines cryptolepine yield
even more than fertilizer application. In comparison, field production of the herb improved
cryptolepine yield better than production in pots, but this did not significantly change the
antiplasmodial activity of cryptolepine extracts, which increased with ageing in the plants.
Based on the present findings, 200 kg/ha of N applied in six equal splits along
with a 9-month growth period is recommended for cultivating the herb. Monitoring the
active ingredient concentration during growth and maturation to ensure that plants are
harvested at the right time with maximum bioactivity should be a major goal of the herbal
industry. From the practical point of view, the increase in root biomass and cryptolepine
yield induced by N application has positive implications since the commercial value of
C. sanguinolenta and farmer income depends on the amount of cryptolepine produced.
Author Contributions: Conceptualization, J.N.A.; methodology, J.N.A., D.O.-S. and R.A.-O.; soft-
ware, B.A.O.; validation, J.N.A. and I.A.-M.; formal analysis, B.A.O.; investigation, F.E.A. and C.M.A.;
resources, C.M.A.; data curation, F.E.A.; writing—original draft preparation, J.N.A. and B.A.O.;
writing—review and editing, J.N.A., B.A.O., F.E.A., D.O.-S., R.A.-O. and I.A.-M.; visualization,
B.A.O.; supervision, J.N.A., D.O.-S. and I.A.-M.; project administration, J.N.A.; funding acquisition,
J.N.A. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Volkswagen Foundation of Germany, grant number (85456).
Plants 2022, 11, 122 14 of 16
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of the University of Ghana, Legon, and approved by the Institutional Review Board
of Noguchi Memorial Institute for Medical Research (NMIMR) (protocol code NMIMR-IRB CPN
028/14–15 and approved on 5 November 2014).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Data included in article/referenced in the article.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Cimanga, K.; De Bruyne, T.; Pieters, L.; Totte, J.; Tona, L.; Kambu, K.; Berghe, D.V.; Vlietinck, A. Antibacterial and antifungal activ-
ities of neocryptolepine, biscryptolepine and cryptoquindoline, alkaloids isolated from Cryptolepis sanguinolenta. Phytomedicine
1998, 5, 209–214. [CrossRef]
2. Dwuma-Badu, D.; Ayim, J.S.; Fiagbe, N.I.; Knapp, J.E.; Schiff, P.L., Jr.; Slatkin, D.J. Constituents of West African medicinal plants
XX: Quindoline from Cryptolepis sanguinolenta. J. Pharm. Sci. 1978, 67, 433–434. [CrossRef]
3. Paulo, A.; Gomes, E.T.; Houghton, P.J. New alkaloids from Cryptolepis sanguinolenta. J. Nat. Prod. 1995, 58, 1485–1491. [CrossRef]
4. Tackie, A.N.; Boye, G.L.; Sharaf, M.H.; Schiff, P.L., Jr.; Crouch, R.C.; Spitzer, T.D.; Johnson, R.L.; Dunn, J.; Minick, D.; Martin,
G.E. Cryptospirolepine, a unique spiro-nonacyclic alkaloid isolated from Cryptolepis sanguinolenta. J. Nat. Prod. 1993, 56, 653–670.
[CrossRef]
5. Addy, M. Cryptolepis: An African traditional medicine that provides hope for malaria victims. HerbalGram 2003, 60, 54–59.
6. Tempesta, M.S. The clinical efficacy of Cryptolepis sanguinolenta in the treatment of malaria. Ghana Med. J. 2010, 44, 1. [PubMed]
7. Borquaye, L.S.; Gasu, E.N.; Ampomah, G.B.; Kyei, L.K.; Amarh, M.A.; Mensah, C.N.; Nartey, D.; Commodore, M.; Adomako,
A.K.; Acheampong, P. Alkaloids from Cryptolepis sanguinolenta as potential inhibitors of SARS-CoV-2 viral proteins: An in silico
study. BioMed Res. Int. 2020, 2020, 1–14. [CrossRef] [PubMed]
8. FDA-Ghana. FDA Approves First Herbal Medicine for the Clinical Trial on COVID-19 Treatment. Food and Drugs Authority
Ghana, Accra, Ghana. 2021. Available online: https://fdaghana.gov.gh/press.php?page=18 (accessed on 24 May 2021).
9. Amissah, J.N.; Gilday, K. Survey on the Importance of Cryptolepis sanguinolenta in the United States of America. Unpublished
work. 2018.
10. Boakye-Yiadom, K. Antimicrobial Properties of Some West African Medicinal Plants II. Antimicrobial Activity of Aqueous
Extracts of Cryptolepis sanguinolenta (Lindl.) Schlechter. Quart. J. Crude Drug Res. 2008, 17, 78–80. [CrossRef]
11. Ansha, C.; Mensah, K. A review of the anticancer potential of the antimalarial herbal Cryptolepis sanguinolenta and its major
alkaloid cryptolepine. Ghana Med. J. 2013, 47, 137–147.
12. Gudivaka, V.R. Synthesis, Analysis and Biological Evaluation of Novel Indoloquinoline Cryptolepine Analogues as Potential
Antitumour Agents. Ph.D. Thesis, Kingston University, London, UK, 2014.
13. Ameyaw, E.O.; Koffuor, G.A.; Asare, K.K.; Konja, D.; Du-bois, A.; Kyei, S.; Forkuo, A.D.; Mensah, R.N.A.O. Cryptolepine, an
indoloquinoline alkaloid, in the management of diabetes mellitus and its associated complications. J. Intercult. Ethnopharmacol.
2016, 5, 263. [CrossRef]
14. Ablordeppey, S.Y.; Fan, P.; Li, S.; Clark, A.M.; Hufford, C.D. Substituted indoloquinolines as new antifungal agents. Bioorganic
Med. Chem. 2002, 10, 1337–1346. [CrossRef]
15. Bamgbose, S.; Noamesi, B. Studies on cryptolepine. Planta Med. 1981, 41, 392–396. [CrossRef] [PubMed]
16. Falconer, J. Non-Timber Forest Products in Southern Ghana; Main Report; Chatham Maritime: Chatham, UK, 1994.
17. Amissah, J.; Osei-Safo, D.; Asare, C.; Missah-Assihene, B.; Danquah, E.; Addae-Mensah, I. Influence of age and staking on the
growth and cryptolepine concentration in cultivated roots of Cryptolepis sanguinolenta (Lindl.) Schlt. J. Med. Plants Res. 2016, 10,
113–121.
18. Monney, M.A.D.; Amissah, N.; Blay, E. Influence of BA and IBA or NAA Combinations on Micropropagation of Cryptolepis
sanguinolenta. Am. J. Plant Sci. 2016, 7, 572–580. [CrossRef]
19. Lubbe, A.; Verpoorte, R. Cultivation of medicinal and aromatic plants for specialty industrial materials. Ind. Crop. Prod. 2011, 34,
785–801. [CrossRef]
20. Stutte, G.W. Process and product: Recirculating hydroponics and bioactive compounds in a controlled environment. HortScience
2006, 41, 526–530. [CrossRef]
21. Erisman, J.W.; Sutton, M.A.; Galloway, J.; Klimont, Z.; Winiwarter, W. How a century of ammonia synthesis changed the world.
Nat. Geosci. 2008, 1, 636–639. [CrossRef]
22. Karamanos, A.J.; Sotiropoulou, D.E. Field studies of nitrogen application on Greek oregano (Origanum vulgare ssp. hirtum (Link)
Ietswaart) essential oil during two cultivation seasons. Ind. Crop. Prod. 2013, 46, 246–252. [CrossRef]
23. Nurzyńska-Wierdak, R. Does mineral fertilization modify essential oil content and chemical composition in medicinal plants.
Acta Sci. Pol.-Hortorum Cultus 2013, 12, 3–16.
24. Gholamhosseinpour, Z.; Hemati, K.; Dorodian, H.; Bashiri-Sadr, Z. Effect of nitrogen fertilizer on yield and amount of alkaloids
in periwinkle and determination of vinblastine and vincristine by HPLC and TLC. Plant Sci. Res. 2011, 3, 4–9.
Plants 2022, 11, 122 15 of 16
25. Ruminska, A.; El Gamal, E.S. Effect of nitrogen fertilization on growth, yield and alkaloid content in Datura innoxia mill.
ActaHortic. 1978, 73, 173–180. [CrossRef]
26. Kapoor, R.; Giri, B.; Mukerji, K.G. Improved growth and essential oil yield and quality in Foeniculum vulgare mill on mycorrhizal
inoculation supplemented with P-fertilizer. Bioresour. Technol. 2004, 93, 307–311. [CrossRef] [PubMed]
27. Rao, B.R. Biomass and essential oil yields of rainfed palmarosa (Cymbopogon martinii (Roxb.) Wats. var. motia Burk.) supplied
with different levels of organic manure and fertilizer nitrogen in semi-arid tropical climate. Ind. Crop. Prod. 2001, 14, 171–178.
[CrossRef]
28. Sifola, M.I.; Barbieri, G. Growth, yield and essential oil content of three cultivars of basil grown under different levels of nitrogen
in the field. Sci. Hortic. 2006, 108, 408–413. [CrossRef]
29. Bloom, A.J.; Frensch, J.; Taylor, A.R. Influence of inorganic nitrogen and pH on the elongation of maize seminal roots. Ann. Bot.
2006, 97, 867–873. [CrossRef]
30. Bown, H.E.; Watt, M.S.; Clinton, P.W.; Mason, E.G. Influence of ammonium and nitrate supply on growth, dry matter partitioning,
N uptake and photosynthetic capacity of Pinus radiata seedlings. Trees 2010, 24, 1097–1107. [CrossRef]
31. El Gendy, A.; El Gohary, A.; Omer, E.; Hendawy, S.; Hussein, M.; Petrova, V.; Stancheva, I. Effect of nitrogen and potassium
fertilizer on herbage and oil yield of chervil plant (Anthriscus cerefolium L.). Ind. Crop. Prod. 2015, 69, 167–174. [CrossRef]
32. Pandey, V.; Patel, A.; Patra, D. Amelioration of mineral nutrition, productivity, antioxidant activity and aroma profile in marigold
(Tagetes minuta L.) with organic and chemical fertilization. Ind. Crop. Prod. 2015, 76, 378–385. [CrossRef]
33. Waraich, E.A.; Ahmad, Z.; Ahmad, R.; Saifullah; Ashraf, M. Foliar applied phosphorous enhanced growth, chlorophyll contents,
gas exchange attributes and PUE in wheat (Triticum aestivum L.). J. Plant Nutr. 2015, 38, 1929–1943. [CrossRef]
34. Zapata, F.; Zaharah, A. Phosphorus availability from phosphate rock and sewage sludge as influenced by the addition of water
soluble phosphate fertilizer. Nutr. Cycl. Agroecosystems 2002, 63, 43–48. [CrossRef]
35. Barku, V.; Opoku-Boahen, Y.; Dzotsi, E. Isolation and pharmacological activities of alkaloids from Cryptolepis sanguinolenta (Lindl.)
schlt. Int. Res. J. Biochem. Bioinform. 2012, 2, 58–61.
36. Kandil, M.A.M.; Khatab, M.E.; Ahmed, S.S.; Schnug, E. Herbal and essential oil yield of Genovese basil (Ocimum basilicum L.)
grown with mineral and organic fertilizer sources in Egypt. J. Für Kult. 2009, 61, 443–449.
37. Ehsanipour, A.; Razmjoo, J.; Zeinali, H. Effect of nitrogen rates on yield and quality of fennel (Foeniculum vulgare Mill.) accessions.
Ind. Crop. Prod. 2012, 35, 121–125. [CrossRef]
38. Habibi, A.; Heidari, G.; Sohrabi, Y.; Badakhshan, H.; Mohammadi, K. Influence of bio, organic and chemical fertilizers on
medicinal pumpkin traits. J. Med. Plants Res. 2011, 5, 5590–5597.
39. Azizi, A.; Yan, F.; Honermeier, B. Herbage yield, essential oil content and composition of three oregano (Origanum vulgare L.)
populations as affected by soil moisture regimes and nitrogen supply. Ind. Crop. Prod. 2009, 29, 554–561. [CrossRef]
40. Abdelaziz, M.E.; Pokluda, R.; Abdelwahab, M.M. Influence of compost, microorganisms and NPK fertilizer upon growth,
chemical composition and essential oil production of Rosmarinus officinalis L. Not. Bot. Horti Agrobot. Cluj-Napoca 2007, 35, 86.
41. Salisbury, F.; Ross, C. Chap. Lipids and aromatic compounds. In Plant Physiology, 2nd ed.; Wadsworth Publishing Co.: Belmont,
CA, USA, 1978.
42. Hatwar, G.; Gondane, S.; Urkade, S. Effect of micronutrients on growth and yield of chilli. J. Soil Crops 2003, 13, 123–125.
43. Ramezani, S.; Rezaei, M.R.; Sotoudehnia, P. Improved growth, yield and essential oil content of basil grown under different levels
of phosphorus sprays in the field. J. Appl. Biol. Sci. 2009, 3, 105–110.
44. Jeshni, M.G.; Mousavinik, M.; Khammari, I.; Rahimi, M. The changes of yield and essential oil components of German Chamomile
(Matricaria recutita L.) under application of phosphorus and zinc fertilizers and drought stress conditions. J. Saudi Soc. Agric. Sci.
2017, 16, 60–65. [CrossRef]
45. Chrysargyris, A.; Panayiotou, C.; Tzortzakis, N. Nitrogen and phosphorus levels affected plant growth, essential oil composition
and antioxidant status of lavender plant (Lavandula angustifolia Mill.). Ind. Crop. Prod. 2016, 83, 577–586. [CrossRef]
46. Baskaran, K.; Srinivas, K.; Kulkarni, R. Two induced macro-mutants of periwinkle with enhanced contents of leaf and root
alkaloids and their inheritance. Ind. Crop. Prod. 2013, 43, 701–703. [CrossRef]
47. Siribel, A.; El Hassan, G.; Muddathir, A.; Alla, A. Effect of Season and Plant Age on Growth and Alkaloids Content of Two Strains
of Catharanthus roseus LG Don. Grown in Sudan. Khartoum Univ. J. Agric. Sci. 2003, 11, 11–27.
48. Sreevalli, Y.; Baskaran, K.; Chandrashekara, R.; Kulkarni, R. Preliminary observations on the effect of irrigation frequency and
genotypes on yield and alkaloid concentration in periwinkle. J. Med. Aromat. Plant Sci. 2000, 22, 356–358.
49. Yousaf, M.; Li, J.; Lu, J.; Ren, T.; Cong, R.; Fahad, S.; Li, X. Effects of fertilization on crop production and nutrient-supplying
capacity under rice-oilseed rape rotation system. Sci. Rep. 2017, 7, 1–9. [CrossRef] [PubMed]
50. Dibb, D.; Thompson, W., Jr. Interaction of potassium with other nutrients. Potassium Agric. 1985, 515–533.
51. Brady, N.C.; Weil, R.R.; Weil, R.R. The Nature and Properties of Soils, 14th ed.; Prentice Hall: Upper Saddle River, NJ, USA, 2008;
Volume 13, pp. 662–710.
52. Ansah, C.; Gooderham, N.J. The popular herbal antimalarial, extract of Cryptolepis sanguinolenta, is potently cytotoxic. Toxicol. Sci.
2002, 70, 245–251. [CrossRef] [PubMed]
53. Laryea, D.; Isaksson, A.; Wright, C.W.; Larsson, R.; Nygren, P. Characterization of the cytotoxic activity of the indoloquinoline
alkaloid cryptolepine in human tumour cell lines and primary cultures of tumour cells from patients. Investig. New Drugs 2009,
27, 402–411. [CrossRef] [PubMed]
Plants 2022, 11, 122 16 of 16
54. Zhu, H.; Gooderham, N.J. Mechanisms of induction of cell cycle arrest and cell death by cryptolepine in human lung adenocarci-
noma a549 cells. Toxicol. Sci. 2006, 91, 132–139. [CrossRef]
55. Owusu-Bennoah, E.; Awadzi, T.; Boateng, E.; Krogh, L.; Breuning-Madsen, H.; Borggaard, O.K. Soil properties of a toposequence
in the moist semi-deciduous forest zone of Ghana. W. Afr. J. Appl. Ecol. 2000, 1, 1–10. [CrossRef]
56. Carballas, T.; Macias, F.; Diaz-Fierros, F.; Carballas, M.; Fernandez-Urrutia, J. FAO-UNESCO soil map of the world. Revised
legend. Inf. Sobre Recur. Mund. De Suelos (FAO) 1990, 60, 146.
57. Page, A.; Miller, R.; Keeney, D. Methods of Soil Analysis, Part 2: Chemical and Microbiological Properties, 2nd ed.; American Society of
Agronomy: Madison, WI, USA, 1982.
58. Bouyoucos, G.J. The hydrometer as a new method for the mechanical analysis of soils. Soil Sci. 1927, 23, 343–354. [CrossRef]
59. Black, C.A. Methods of soil analysis. Part 2. In Chemical and Microbiological Properties; American Society of Agronomy: Madison,
WI, USA, 1965.
60. ISO. ISO 11261: Soil Quality Determination of Total Nitrogen-Modified Kjeldahl Method; International Organization for Standardization:
Geneva, Switzerland, 1995.
61. ISO. ISO 14263: Soil Quality Determination of Phosphorus-Spectrometric Determination of Phosphorus Soluble in Sodium Hydrogen
Carbonate Solution; International Standard Organisation: Geneva, Switzerland, 1994.
62. Chapman, H.; Pratt, P. Methods of analysis for soils, plants and waters. Soil Sci. 1961, 93, 169–176. [CrossRef]
63. Trager, W.; Jensen, J.B. Human malaria parasites in continuous culture. Science 1976, 193, 673–675. [CrossRef] [PubMed]
64. Bennett, T.N.; Paguio, M.; Gligorijevic, B.; Seudieu, C.; Kosar, A.D.; Davidson, E.; Roepe, P.D. Novel, rapid, and inexpensive
cell-based quantification of antimalarial drug efficacy. Antimicrob. Agents Chemother. 2004, 48, 1807–1810. [CrossRef] [PubMed]
65. Ayisi, N.K.; Appiah-Opong, R.; Gyan, B.; Bugyei, K.; Ekuban, F. Plasmodium falciparum: Assessment of selectivity of action of
chloroquine, Alchornea cordifolia, Ficus polita, and other drugs by a tetrazolium-based colorimetric assay. Malar. Res. Treat. 2011,
2011, 1–7. [CrossRef] [PubMed]
66. Appiah-Opong, R.; Asante, I.K.; Safo, D.O.; Tuffour, I.; Ofori-attah, E.; Uto, T.; Nyarko, A.K. Cytotoxic effects of Albizia zygia
(DC) JF Macbr, a Ghanaian medicinal plant, against human T-lymphoblast-like leukemia, prostate and breast cancer cell lines. Int.
J. Pharm. Pharm. Sci. 2016, 8, 392–396.
67. VSN. GenStat Edition 12.1 for Windows. VSN International Ltd.; UK, 2009. Available online: https://www.vsni.co.uk/software/
genstat/ (accessed on 27 November 2021).