GC–MS and LC-TOF–MS profiles, toxicity, and macrophage-dependent in vitro anti-osteoporosis activity of Prunus africana (Hook f.) Kalkman Bark

Abstract

Abstract Osteoporosis affects millions of people worldwide. As such, this study assessed the macrophage-dependent in vitro anti-osteoporosis, phytochemical profile and hepatotoxicity effects in zebrafish larvae of the stem bark extracts of P. africana. Mouse bone marrow macrophages (BMM) cells were plated in 96-well plates and treated with P. africana methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 µg/ml for 24 h. The osteoclast tartrate-resistant acid phosphatase (TRAP) activity and cell viability were measured. Lipopolysaccharides (LPS) induced Nitrite (NO) and interleukin-6 (IL-6) production inhibitory effects of P. africana bark extracts (Methanolic, 150 µg/ml) and β-sitosterol (100 µM) were conducted using RAW 264.7 cells. Additionally, inhibition of IL-1β secretion and TRAP activity were determined for chlorogenic acid, catechin, naringenin and β-sitosterol. For toxicity study, zebrafish larvae were exposed to different concentrations of 25, 50, 100, and 200 µg/ml P. africana methanolic, ethanolic and water bark extracts. Dimethyl sulfoxide (0.05%) was used as a negative control and tamoxifen (5 µM) and dexamethasone (40 µM or 80 µM) were positive controls. The methanolic P. africana extracts significantly inhibited (p

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GC–MS
and LC‑TOF–MS proles, toxicity,
and macrophage‑dependent
in vitro anti‑osteoporosis activity
of Prunus africana (Hook f.)
Kalkman Bark
Richard Komakech1,2,5, Ki‑Shuk Shim3, Nam‑Hui Yim4, Jun Ho Song1, Sungyu Yang1,
Goya Choi1, Jun Lee1,2, Yong‑goo Kim1, Francis Omujal5, Denis Okello1,2,
Moses Solomon Agwaya5, Grace Nambatya Kyeyune5, Hyemin Kan6, Kyu‑Seok Hwang6,
Motlalepula Gilbert Matsabisa7 & Youngmin Kang1,2*
Osteoporosis aects millions of people worldwide. As such, this study assessed the macrophage‑
dependent in vitro anti‑osteoporosis, phytochemical prole and hepatotoxicity eects in zebrash
larvae of the stem bark extracts of P. africana. Mouse bone marrow macrophages (BMM) cells were
plated in 96‑well plates and treated with P. africana methanolic bark extracts at concentrations of
0, 6.25, 12.5, 25, and 50 µg/ml for 24 h. The osteoclast tartrate‑resistant acid phosphatase (TRAP)
activity and cell viability were measured. Lipopolysaccharides (LPS) induced Nitrite (NO) and
interleukin‑6 (IL‑6) production inhibitory eects of P. africana bark extracts (Methanolic, 150 µg/
ml) and β‑sitosterol (100 µM) were conducted using RAW 264.7 cells. Additionally, inhibition of
IL‑1β secretion and TRAP activity were determined for chlorogenic acid, catechin, naringenin and
β‑sitosterol. For toxicity study, zebrash larvae were exposed to dierent concentrations of 25, 50,
100, and 200 µg/ml P. africana methanolic, ethanolic and water bark extracts. Dimethyl sulfoxide
(0.05%) was used as a negative control and tamoxifen (5 µM) and dexamethasone (40 µM or 80 µM)
were positive controls. The methanolic P. africana extracts signicantly inhibited (p < 0.001) TRAP
activity at all concentrations and at 12.5 and 25 µg/ml, the extract exhibited signicant (p < 0.05) BMM
cell viability. NO production was signicantly inhibited (all p < 0.0001) by the sample. IL‑6 secretion
was signicantly inhibited by P. africana methanolic extract (p < 0.0001) and β‑sitosterol (p < 0.0001)
and further, chlorogenic acid and naringenin remarkably inhibited IL‑1β production. The P. africana
methanolic extract signicantly inhibited RANKL‑induced TRAP activity. The phytochemical study of
P. africana stem bark revealed a number of chemical compounds with anti‑osteoporosis activity. There
was no observed hepatocyte apoptosis in the liver of zebrash larvae. In conclusion, the stem bark of
P. africana is non‑toxic to the liver and its inhibition of TRAP activity makes it an important source for
future anti‑osteoporosis drug development.
OPEN
1Herbal Medicine Resources Research Center, Korea Institute of Oriental Medicine (KIOM), 111 Geonjae-ro,
Naju-si, Jeollanam-do 58245, Republic of Korea. 2University of Science and Technology (UST), Korean
Convergence Medicine Major, KIOM campus, 1672 Yuseongdae-ro, Yuseong-gu, Daejeon 34054, Republic of
Korea. 3Korea Institute of Oriental Medicine (KIOM), 1672 Yuseongdae-ro, Yuseong-gu, Daejeon 34054, Republic
of Korea. 4Korean Medicine Application Center, Korea Institute of Oriental Medicine, 70 Cheomdan-ro, Dong-gu,
Daegu 41062, Republic of Korea. 5Natural Chemotherapeutics Research Institute (NCRI), Ministry of Health,
P.O. Box 4864, Kampala, Uganda. 6Bio and Drug Discovery Division, Korea Research Institute of Chemical
Technology, Daejeon, Republic of Korea. 7IKS Research Group, Department of Pharmacology, Faculty of Health
Sciences, University of the Free State, Bloemfontein 9301, Free State, South Africa. *email: ymkang@kiom.re.kr
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Osteoporosis is a silent but one of the major global health problems characterized by deterioration of bone micro-
architecture and low bone mass1,2. It is one of the leading causes of morbidity in older people above 40 years3. is
condition occurs when the rate of bone resorption is higher than the rate of bone formation and consequently
presents a greater risk of fractures for the persons suering from it. Factors such as aging, sex steroid deciency,
and as well as menopause in women have been associated with a higher risk of osteoporosis4. Activated mac-
rophages have also been implicated in the pathogenesis of osteoporosis by stimulating the development of osteo-
clastogenesis-associated bone loss5. Cytokines such as tumor necrosis factor alpha (TNFα), interleukin-1 (IL-1β)
and interleukin-6 (IL-6) are pro-inammatory and play central role in inammation of which IL-6 is the most
important in chronic inammatory and autoimmune diseases, cytokine storm and cancer6,7. IL-6 is fundamental
in a number of processes including bone metabolism, inammation, hematopoiesis7. IL-6 is also implicated in
mediation of IL-1 eects, a potent bone resorption stimulator8. Although a number of conventional methods
have been employed in the treatment of osteoporosis including bisphophates and estrogen hormonal therapy,
adverse eects associated with these therapies that are reported to limit their use include; gastrointestinal tract
disturbances and burning sensation9. Exploration of other avenues including the use of natural products in the
treatment of osteoporosis have been suggested to oer a better alternative with lesser adverse side eects9. Herbal
medicines have been used over the years to prevent and treat osteoporosis condition2. e anti-osteoporosis of
herbal products has been attributed to the secondary metabolites including alkaloids, terpenes, steroids, and
phenolic compounds10,11 and these have led to the development of a number of drugs over the years with great
therapeutic activities12. Prunus africana (Hook f.) Kalkman (Family Rosaceae), commonly called African cherry
is an evergreen plant endemic in sub-Saharan Africa13, and contains a number of secondary metabolites like
terpenes, alkaloids, phenolic compounds, and sterols in its stem bark14. For centuries, P. africana has been used
in Africa to treat myriad of diseases including prostate cancer, hyperplasia, diabetes, malaria, and inammatory
conditions15. Despite its immense medicinal uses and with a wide array of secondary metabolites, P. africana
has not been investigated for anti-osteoporosis activity yet it is used by the persons with prostate cancer who are
vulnerable to suer from osteoporosis16,17. Hence, this study evaluated the phytochemistry P. africana bark extract
and it’s in vitro anti-osteoporosis activity based on osteoclast tartrate-resistant acid phosphatase (TRAP) as a
cytochemical marker of osteoclasts. However, due to the toxicity associated with some of the herbal medicines
such as liver damage18 and owing to association of liver health and osteoporosis19, this study also evaluated the
hepatotoxicity of P. africana bark extracts in zebrash (Danio rerio) larvae.
Materials and methods
Plant material and preparation of extract. e study was conducted in accordance to the relevant
institutional, national, and international guidelines and legislation. e stem bark of P. africana (1 kg) was
obtained from P. africana tree (Fig.1a,b) in the herbal garden of Natural Chemotherapeutics Research Institute,
Ministry of Health, Uganda. e study material was identied by Dr. Sungyu Yang at Korea Institute of Oriental
medicine (KIOM) and a voucher specimen (number KIOM201901022377) of the sample was deposited in the
Korean Herbarium of Standard Herbal Resources (Index Herbarium code: KIOM) at KIOM, South Korea. e
stem bark (Fig.1c) was dried in an oven at 40°C and then ground using a steel pulverizing machine (250G
New Type Pulverizing Machine, Model RT-N04-2V, Taiwan) to obtain a ne powder (Fig.1d). 500g of the
ne powder sample was extracted by maceration using 1,500ml of methanol. e extract was ltered using
Whatman lter No. 1 aer 24h. and concentrated under a vacuum reduced pressure at 40°C, 70rpm, using an
EYELA N-1200B (Tokyo Rikakikai Co. Ltd, Japan) ecient rotary evaporator. e concentrated extract was then
vacuum dried and yielded 60g of extract. e resultant dried extract was then used in the subsequent TRAP
assay, cell viability assay, and experiments on the production of inammatory factors.
Gas chromatography‑mass spectrometry (GC–MS) sample preparation and analysis. e P.
africana bark sample was extracted in 100% methanol by sonication for 30min. e extract was then prepared
at 50µg/L; ltered through a 0.2μm syringe membrane lter from Whatman Ltd (Maidstone, UK) and subjected
to GC–MS analysis. e analysis was performed in a 7890B GC–MS system (Agilent Technologies, Atlanta, GA,
USA), coupled with a 7977B model mass detector (Agilent Technologies, Atlanta, GA, USA) using DB-5 MS
capillary column (30m × 0.25mm × 0.25μm). Chromatographic conditions were as follows: the extract (1μL)
was injected in split mode with a ratio of 1/20 at 250°C; oven initial temperature was 50°C and increased 110°C
during 5min, followed by heating at a rate of 7°C/min at 300°C. e mass analyzer was set to scan from 30 to
600 amu. Peak identication was carried out by comparison of the experimental mass spectrum in the National
Institute of Standards and Technology (NIST) and Wiley GC–MS libraries.
Liquid chromatography time‑of ight mass spectrometry (LC‑TOF–MS) analysis. e P. af ri-
cana bark sample was extracted and prepared as in “Gas chromatography-mass spectrometry (GC–MS) sample
preparation and analysis” above. e LC-TOF–MS analysis was performed on an Agilent 1290 innity II system
coupled with an AB SCIEX Triple TOF 5600 mass spectrometer equipped with electrospray ionization. Gemini®
C18 (250mm × 4.6mm i.d., 5μm, Phenomenex, USA) was used for column separation. e column temperature
was maintained at 40°C, the ow rate was 1.0ml/min, and the injection volume was 10μl. e optimal mobile
phase consisted of a linear gradient system of (A) 0.1% formic acid in water and (B) 0.1% formic acid in ace-
tonitrile, 0–2min, 3% B; 2–30min, 3–35% B; 30–31min, 35–50% B; 31–35min, maintained 50% B; 35–40min,
100% B; 40–45min, maintained 100% B. Positive mode was applied in the ESI source with the following param-
eters: gas 1 = 50 psi, gas 2 = 50 psi, temperature = 500°C, and 5500V ion spray voltage with 30 psi curtain gas.
Intact protonated molecular ions [M-Na]+ were detected via TOF–MS scan (100 psi declustering potential, 10V
collision energy, 100–2000Da TOF MS scan range, and 250ms accumulation time). Negative mode was applied
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in the ESI source with the following parameters: gas 1 = 50 psi, gas 2 = 50 psi, temperature = 500°C, and − 4500V
ion spray voltage with 30 psi curtain gas. Intact protonated molecular ions [M-H]− were detected via TOF–MS
scan (− 100 psi declustering potential, − 10V collision energy, 100–2000Da TOF MS scan range, and 250ms
accumulation time).
Inhibitory eect of P. africana on osteoclastogenesis. Cell culture and authentication. Mouse bone
marrow macrophages (BMMs) were isolated from the tibia and femur of mice (male ICR mouse, 7weeks old) by
ushing with PBS as describe in previous study20. Aer over-night incubation in non-coated culture dish, non-
attached cells, which were regarded as BMMs, were collected and cultured in proliferation medium [α-MEM
medium with 10% FBS and macrophage-colony stimulating factor (M-CSF) (60ng/mL)] for 7days. To dier-
entiate osteoclasts, BMMs (1 × 104 cells/well, 96-well plates) were cultured in α-MEM medium containing 10%
FBS, M-CSF (60ng/mL), and RANKL (100ng/mL) for 4days.
TRAP assay and BMM cell viability. is was performed following the previously described method21. e
measurement of osteoclast TRAP activity was based on the generation of absorbance by incubating BMM cells
with TRAP buer (50mM sodium tartrate, 0.12M sodium acetate, pH 5.2) and p-nitrophenyl phosphate (1mg/
ml) for 15min. For TRAP staining, the BMM cells were incubated with TRAP buer containing naphthol
AS-MX phosphate (0.1mg/ml) and Fast Red Violet (0.5mg/ml). e BMM cells were then cultured with P.
africana methanolic extract at 0, 6.25, 12.5, 25, and 50µg/ml concentrations and 50μM of β-sitosterol, chloro-
genic acid, catechin and naringenin each in the presence of RANKL for 6days. e osteoclast TRAP activity was
determined using a colorimetric assay with p-nitrophenyl phosphate as a substrate. e cell viability was deter-
Figure1. Prunus africana medicinal plant. (a) P. africana tree. (b) Stem of P. africana with part of its bark
harvested for medicine purpose. (c) Harvested and dried P. africana stem bark. (d) Pulverized P. africana stem
bark.
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mined using Cell Counting Kit-8 (CCK) (WST-8/CCK8; Dojindo), according to the manufacturer’s instructions.
For the measurement of cell viability, cells were plated in 96-well plates and treated with P. africana extracts
concentrations as above for 24h. Aer incubating with the CCK solutions and the cells for 1h, the absorbance
was measured at 450nm using a microplate reader (Versa Max) and results were presented as a percentage of
the vehicle control.
Nitrite (NO) assay and RAW 264.7 cell viability. Murine macrophage RAW 264.7 cells were cultured
in DMEM medium supplemented with 100 U/mL of penicillin, 100 ug/mL of streptomycin, and 10% heat-inac-
tivated FBS. e nitrite concentration in the supernatant from cultured cells was analyzed using the Griess reac-
tion test. RAW 264.7 cells were plated at a density of 5 × 104 cells/mL in 96-well culture plates, pre-incubated with
samples (P. africana methanolic extracts (150μg/mL), β-sitosterol, chlorogenic acid, catechin and naringenin
(100μM each) for 3h, and stimulated with LPS (200ng/ml) for 24h. Griess reagent (1% sulfanilamide, 0.1%
N-1-napthylethylenediamine dihydrochloride, and 2.5% phosphoric acid) was mixed with an equal volume of
cell supernatant, and absorbance was measured at 570nm using the ELISA reader. Sodium nitrite was used as a
standard. Dexamethasone was used as a positive control (40 or 80μM).
Enzyme‑linked immunosorbent assay (ELISA). e concentrations of the inammatory cytokines
IL-1β (R&D, USA) and IL-6 (MyBiosource, USA) in culture supernatant was determined using ELISA antibody
kits following the manufacturer1s protocol (MyBiosource, USA). RAW264.7 cells were grown in 96-well culture
plates at a density of 5 × 104 cells/mL, pre-incubated with samples for 3h, and stimulated with LPS for 6h (IL-
1β) or 24h (IL-6). e cytokines produced in each sample were calculated from standard curves using known
concentrations of recombinant cytokines for each ELISA antibody kit.
Hepatotoxicity assay in zebrash (Danio rerio) larvae. is was performed following the previously
described method21. All methods were carried out in accordance with relevant guidelines and regulations. All
experimental protocols were approved by Korea Research Institute of Chemical Technology research ethics com-
mittee and conducted in compliance with the ARRIVE guidelines. Zebrash larvae were used for this study as
previously described22. At 96h post-fertilization (hpf), the larvae were transferred to a transparent 24-well plate
(N = 10/well) with 1ml of embryonic medium. e larvae were then exposed to increasing concentrations of 25,
50, 100, and 200µg/ml of P. africana methanolic and ethanolic extracts and water extract from 90 to 120 hpf.
Dimethyl sulfoxide (DMSO) was used as a negative control while 5μM of tamoxifen (Sigma-Aldrich, St. Louis,
MO, USA) was used as a positive control. To obtain images, the larvae were anesthetized in tricaine (Sigma-
Aldrich), mounted in 3% methyl cellulose (Sigma-Aldrich), and observed under a Leica MZ10 F stereomicro-
scope equipped with a Leica DFC425 camera and Leica application Suite soware (version 4.5).
Statistical analysis. Data were represented as the mean ± standard deviation. Statistical signicance
between groups was analyzed using the Student’s t-test and p-values < 0.05 were considered statistically signi-
cant.
Results
GC/MS analysis. e GC/MS analysis of the P. africana extract was based on mass spectra, retention times,
and quality ratio analysis revealed the presence of 32 components (Table1) including 3-Furanmethanol (1),
Dihydroxyacetone (2), Benzoic acid, methyl ester (3), 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-
(4), benzoic acid (5), Catechol (6), 4-Vinylphenol (7), 5-Hydroxymethyl-2-furaldhyde (8), Isosorbide (9),
Phenol, 2,6-dimethoxy- (10), 4-Hydroxy-3-methoxybenzaldehyde (11), 3,4-Altrosan (12), Mandelamide (13),
Vanillic acid (14), Benzenepropanol, 4-hydroxy-3-methoxy- (15), Benzaldehyde, 4-hydroxy-3,5-dimethoxy-
(16), 4-(hydroxymethyl)-2,6-dimethoxyphenol (17), (E)-4-(3-Hydroxyprop-1-en-1-yl)-2-methoxyphenol (18),
6-Hydroxy-5-triuoromethylcyclohexa-1,3-diene (19), Benzoic acid, 4-hydroxy-3,5-dimethoxy- (20), Isopro-
pyl myristate (21), Sorbitol (22), n-Hexadecanoic acid (23), 9,12-Octadecadienoic acid (Z,Z)- (24), Oleic acid
(25), Octadecanoic acid (26), Benzyl, beta-d-glucoside (27), 9-Octadecenamide, (Z)- (28), (R)-alpha-(beta-D-
glucopyranosyloxy)benzene-acetonitrile (29), 13-Docosenamide, (Z)- (30), Squalene (31), Beta-Sitosterol (32).
LC‑TOF–MS analysis. Based on the chemical proling by LC-TOF–MS analysis, the various phytochemi-
cals were detected from the P. africana extract. In the positive ion mode, as a result of analysis using retention
index libraries, 65 peaks with a library score over 90% were identied from the P. africana extract (Supple-
mentary TableY). Among them, 24 components showed reliable mass (over 98% of library score) in the P.
africana extract, especially, 7 components (Astragalin, Chlorogenic acid, Coproporphyrin I, Hyperin, Luteolo-
side, Mesoporphyrin IX and Naringenin), showed the accurate mass according to result of 100% library score
(Table2). In the negative ion mode, 72 peaks with a library score over 90% were identied from the P. africana
extract (Supplementary TableZ). Among them, 29 components showed the reliable mass (over 98% of library
score) in the P. africana extract, especially, 5 components (Pedunculoside, Luteoloside, Hexadecanedioic acid,
Guanosin, Betulonic acid and Naringenin), showed the accurate mass according to result of 100% library score
(Table2). In the present study, among the identied primary and secondary metabolic components, Catechin
showed the largest peak area among the identied primary and secondary metabolic components in the P. af ri-
cana extract (Supplementary TableY and Z).
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Table 1. Phytochemical components identied in the stem bark of Prunus africana methanolic extract by GC/
MS analysis.
Peak no Identied compound tR (min) % of total Quality (%)
1 3-Furanmethanol 5.96 0.39 93
2 Dihydroxyacetone 7.01 2.22 74
3 Benzoic acid, methyl ester 12.83 0.46 91
4 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- 13.95 1.13 95
5 benzoic acid 14.68 14.02 91
6Catechol 15.17 4.74 91
7 4-Vinylphenol 15.61 0.77 72
8 5-Hydroxymethyl-2-furaldhyde 15.81 0.31 89
9 Isosorbide 17.10 0.98 93
10 Phenol, 2,6-dimethoxy- 18.39 0.41 94
11 4-Hydroxy-3-methoxybenzaldehyde 19.30 0.41 93
12 3,4-Altrosan 20.84 3.30 76
13 Mandelamide 21.54 0.40 93
14 Vanillic acid 22.13 0.93 95
15 Benzenepropanol, 4-hydroxy-3-methoxy- 23.61 2.14 92
16 Benzaldehyde, 4-hydroxy-3,5-dimethoxy- 23.83 0.75 91
17 4-(hydroxymethyl)-2,6-dimethoxyphenol 24.57 0.17 87
18 (E)-4-(3-Hydroxyprop-1-en-1-yl)-2-methoxyphenol 25.03 0.18 91
19 6-Hydroxy-5-triuoromethylcyclohexa-1,3-diene 25.98 3.65 59
20 Benzoic acid, 4-hydroxy-3,5-dimethoxy- 26.13 0.29 96
21 Isopropyl myristate 26.31 0.40 99
22 Sorbitol 27.95 0.05 91
23 n-Hexadecanoic acid 28.21 4.95 99
24 9,12-Octadecadienoic acid (Z,Z)- 30.51 0.20 99
25 Oleic acid 30.58 0.76 98
26 Octadecanoic acid 30.87 0.47 97
27 Benzyl, beta-d-glucoside 31.90 0.32 87
28 9-Octadecenamide, (Z)- 33.35 1.95 99
29 (R)-alpha-(beta-D-glucopyranosyloxy) benzene-acetonitrile 35.10 6.60 83
30 13-Docosenamide, (Z)- 37.88 6.49 99
31 Squalene 38.38 1.09 99
32 Beta-Sitosterol 43.55 8.37 99
Table 2. Phytochemical components identied in the stem bark of P. africana methanolic extract by LC-TOF–
MS analysis.
No Name Mass ([M-Na]+) Founded mass RT (min) Founded RT (min) Peak area Library score (%)
1 Astragalin 449.1083 449.1083 23.25 23.22 1129.49 100.00
2 Chlorogenic acid 355.1024 355.1024 13.16 13.14 2289.52 100.00
3Coproporphyrin I 655.4932 655.4961 1.19 1.32 1974.83 100.00
4 Hyperin 465.1033 465.1033 23.12 23.12 3329.70 100.00
5 Luteoloside 449.1787 449.1787 22.56 22.56 4448.29 100.00
6 Mesoporphyrin IX 567.2814 567.2809 42.57 42.56 1217.89 100.00
7 Naringenin 273.0762 273.0762 33.74 33.73 6559.30 100.00
No Name Mass ([M-H]–) Founded mass RT (min) Founded RT (min) Peak area Library score (%)
1Pedunculo-
side + HCOOH 695.3693 695.3696 34.39 34.38 20,498.21 100.00
2 Luteoloside 447.0696 447.0695 23.18 23.21 1513.48 100.00
3 Hexadecanedioic acid 285.1910 285.1909 40.49 40.48 4854.99 100.00
4 Guanosine 282.0683 282.0684 6.66 6.65 7969.05 100.00
5 Betulonic acid 453.2775 453.2776 42.97 42.97 1209.40 100.00
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TRAP assay and BMM viability. TRAP activity was signicantly (p < 0.001) inhibited compared to the
control at concentrations of 6.25, 12.5, 25, and 50µg/ml methanolic P. africana stem bark extracts (Fig.2A).
Prunus africana methanolic stem bark extract had a signicant (p < 0.05) simulative eect at concentrations
of 12.5 and 25µg/ml on the cell viability of BMM cells (Fig.2B) compared to the control. However, at a higher
concentration of 50µg/ml the methanolic stem bark extract, the cell viability reduced to 70% compared to the
control. e high viability of the BMM cells may indicate the non-cytotoxicity of P. africana bark.
e eects of β-sitosterol, chlorogenic acid, catechin and naringenin (50μM each) on RANKL-induced TRAP
activity representing osteoclastogenesis were evaluated. We found that these compounds signicantly inhibited
RANKL-induced TRAP activity without showing cell toxicity (Fig.3).
Inhibitory eect on NO production. Since NO production is correlated with various inammatory dis-
eases, we determined the suppressive eects of samples (P. africana (PA) methanolic extract, β-sitosterol, chlo-
rogenic acid, catechin and naringenin) on NO levels in RAW264.7 cells via LPS stimulation. To determine NO
levels in the supernatant, cells were pre-treated with samples for 3h, followed by stimulation with LPS for 24h,
and then measured using Griess reagent. As the positive control, dexamethasone showed strong suppressive
eect on NO secretion upon LPS stimulation. All the investigated samples dramatically inhibited NO produc-
tion aer LPS stimulation (Fig.3b). All samples did not signicantly aect cell viability and β-sitosterol that
increased it (Fig.3a).
Inhibitory eect on IL‑6 and IL‑1β levels. e eects of samples on inammatory cytokine, IL-6, secre-
tion in macrophages were evaluated using enzyme-linked immunosorbent assay (ELISA). IL-6 secretion was
signicantly inhibited by PA-methanol (p < 0.001) and β-sitosterol (p < 0.0001) (Fig.3c). Chlorogenic acid and
naringenin, but not catechin, signicantly inhibited LPS-induced IL-1β level as shown in Fig.3d.
Hepatotoxicity in zebrash larvae. In this study, the zebrash larvae exposure was done from 96 to 120
hpf and those exposed to DMSO showed no liver cell death (shown by white dash line) (Fig.4A) but tamoxifen
treatment resulted in liver cell death (shown by red dash line) (Fig.4B). At a concentration of 50 and 100µg/
ml water extract of P. africana, 30% and 10% of the zebrash larvae survived at 120 hpf respectively and death
of hepatocytes was not observed in them (Fig.4C). However, at a higher concentration of 200µg/ml P. a fr i-
cana water extract, 100% larvae mortality was observed before 120 hpf. At a concentration of 25µg/ml etha-
nolic extract, 50% of the zebrash larvae survived and hepatocytes death was not observed in them at 120 hpf
(Fig.4D). However, at higher concentrations of 50 and 100µg/ml, 100% larvae mortality rate was observed at
120 hpf. 100% larvae mortality was observed for larvae exposed to P. africana bark methanolic extracts at various
concentrations of 25, 50, and 100µg/ml before the 120 hpf.
Discussion
TRAP is a specic and reliable cytochemical marker used as a measure of activated macrophages23. e bone
marrow macrophages play a major role in the activation and formation of osteoclastsand hence important for
the pathogenesis of osteoporosis5. Osteoporosis reects increased osteoclast function relative to that of osteo-
blasts and hence the pharmacological arrest of osteoclasts is a mainstay in the treatment of systemic bone loss24.
TRAP activity is an important cytochemical marker of osteoclasts and its concentration in the serum is utilized
as a biochemical and histochemical marker of osteoclast function and degree of bone resorption25. erefore,
as a measure of osteoclast number and bone resorption, TRAP plays a vital role in osteoporosis diagnosis and
prognosis23. And as evidenced in the previous study, the suppressing of TRAP pathway prevented ovariectomy-
induced osteoporosis in vivo26. erefore, the signicant (p < 0.001) inhibition compared to the control at con-
centrations of 6.25, 12.5, 25, and 50µg/ml methanolic P. africana stem bark extracts is an indication of P. africana
anti-osteoporosis eects. Additionally, chlorogenic acid, catechin, naringenin and β-sitosterol that are present
Figure2. Eect of P. africana on TRAP activity in BMM. e BMM were cultured with P. africana bark extracts
in the presence of RANKL for 6days and TRAP activity of osteoclasts measured by colorimetric assay using
p-nitrophenyl phosphate as a substrate. Cell viability was determined using Cell Counting Kit-8 following
manufacturer’s instruction. *p < 0.05 and ***p < 0.001.
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in P. africana stem bark have been demonstrated in our study to remarkably inhibit RANKL-induced TRAP
activity explaining indeed the antiosteoporotic potential of the plant stem bark.
e anti-osteoporosis activity of P. africana bark extracts may be attributed to some of the compounds present
in it including astragalin, hyperin, luteoloside, mesoporphyrin, naringenin, chlorogenic acid, β-sitosterol and
catechin (Tables1 and 2, and supplementary TablesYand Z). Additionally, previous studies have also showed
that P. africana stem bark is indeed rich in these compounds with anti-osteoporosis activity (Table3). Astragalin
demonstrated estrogenic anti-osteoporosis activity and signicantly increased proliferation in osteoblastic cells
(UMR-106)27. In another study, Astragalin promoted dierentiation in MC3T3-E1 osteoblastic cells through
activation of MAPK and BMP pathways and promoted invivo bone formation28. Further, a number of other
studies have showed anti-osteoporosis activity of Astragalin10,29,30, and its presence in P. africana stem bark as
conrmed in our study shows that it indeed contributes to the anti-osteoporosis activity of the plant stem bark.
Hyperin was one of the major 3 chemical compounds in Cuscuta chinensis that showed anti-osteoporosis eects29.
In fact in their study, Tao, etal.29 ascribed the anti-osteoporosis eect of C. chinensis to hyperin as a result of its
high positive correlation to anti-osteoporosis activity. Hyperin was also reported to markedly increase alkaline
phosphatase (ALP) activity in osteoblast cells31.
Luteoloside, a natural compound is known to suppress activity of osteoclasts thus could potentially be used
for treating bone metabolism disorders including osteoporosis32. Luteoloside in a previous study was showed
to possess strong inhibition against LPS induced osteolysis in an invivo study32. Further, it was also dem-
onstrated to suppress dierentiation of RANKL-induced osteoclast and decrease bone resorption tendency
dose dependently32. e anti-osteoclastic and anti-resorptive actions of luteoloside were not only through
blockage of NFATc1 activity and debilitation of RANKL-mediated Ca2+signaling but also through MAPK and
NF-κB pathways32. Mesoporphyrin, a porphyrin derivative has been reported to possess anti-inammatory
activity through inhibiting IL-6 production33. Since IL-6 potently activates osteoclasts and is responsible for
bone resorption34, mesoporphyrin IX may thus possess anti-osteoporosis activity through inhibition of IL-6
production.
Figure3. Eects of samples (PA-methanol, β-sitosterol, chlorogenic acid, catechin, and naringenin) on (a)
cell viability, (b-d) the production of inammatory factors (nitric oxide, IL-6, or IL-1β), or (e) TRAP activity.
Aer 3h pre-incubation of samples, RAW 264.7 cells were treated with LPS for 6 to 48h depending on the
assay condition. (a) Cell viability was measured using a CCK assay. (b) Nitric oxide content in the medium was
determined using Griess reagent assay; (c) IL-6 and (d) IL-1β cytokine levels in the medium were measured
using ELISA kit. (e) TRAP activity was examined by using TRAP buer containing naphthol AS-MX phosphate.
Positive control: 40 or 80μM dexamethasone. As a control, cells were incubated with the vehicle alone. *
p < 0.05, ** p < 0.01 and *** p < 0.001.
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Naringenin has been reported to signicantly inhibit osteoclastogenesis with inhibitions of up to 96 ± 1%
at 50μg/ml35. Naringenin has also been demonstrated to not only signicantly inhibit secretion of monocyte
chemoattractant protein-1, interleukin (IL)-1α and IL-23 but also markedly decrease release of a bone resorp-
tion activity indicator, helical peptide 620–633, thus greatly inhibiting osteoclastic bone resorption and human
osteoclastogenesis35. La, etal.35 indicates that naringenin could be used to treat bone-related diseases such as
Table 3. Summary of the previous studies on anti-osteoporosis eects of some of the compounds identied in
P. africana.
S/No Phytochemical compounds Compound structure Anti-osteoporosis eects Reference
(a) Vanillic acid
Improves bone mineral density and bone mineral content; Protects the
trabecular structure from being degraded by osteoclasts 43,44
(b) Sorbitol
Retards bone resorption 45
(c) Octadecanoic acid (Stearic acid)
Inhibits osteoclastogenesis invitro 46
(d) β-sitosterol
Inhibits osteoporosis through retardation of acute inammation 47
Figure4. Hepatotoxicity assay in zebrash larvae at 120 hpf. (A) DMSO as a negative control did not induce
hepatotoxicity. (B) Tamoxifen induced liver cell death (shown by red arrow). (C) 100µg/ml P. africana water did
not induce hepatotoxicity. (D) 25µg/ml P. africana ethanol did not induce hepatotoxicity.
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osteoporosis. In the current study, naringenin was observed to signicantly inhibit LPS-induced NO production
to a level even greater than the eect of dexamethasone (positive control) and also remarkably suppress IL-1β
generation. Since increased NO level inhibits growth and dierentiation of osteoblasts36, and IL-1β stimulates
bone resorption37, naringenin suppression of NO and IL-1β production signies that it is important in prevent-
ing osteoporosis. In another study, naringenin was showed to signicantly promote osteogenic dierentiation38.
Naringenin being present in the stem bark of P. africana contributes to its anti-osteoporosis activity. Catechin
was identied as possessing the strongest osteogenic eects from a batch of herbal ingredients used in traditional
Chinese medicine using human mesenchymal stem cells (hMSCs)39. Catechin was reported to increase the activ-
ity of ALP, deposition of calcium and Runx2 mRNA expression among others39. It was thus proposed to enhance
osteogenesis through increasing protein phosphatases 2A (PP2A) level inhibiting extracellular signal-regulated
kinase (ERK) signaling in hMSCs39. In another study, catechin rich extract was demonstrated to promote for-
mation and enhance survival of osteoblasts and inhibiting the activity and growth of osteoclasts40. Similar to
our ndings, catechin in previous studies has been documented to suppress NO production in LPS stimulated
macrophages41,42. us, our study suggests that catechin exerts antiosteroporotic eect by eliminating the inhibi-
tory eect of NO on growth and dierentitation of osteoblasts. e presence of catechin in the stem bark of P.
africana enhances its anti-osteoporosis potential.
Vanillic acid has been reported to exhibit anti-osteoporotic activity by inhibitory eects on bone resorption48;
improving bone mineral density and bone mineral contentand as well as biomechanical stability43 and protect-
ing trabecular structure from degradation by osteoclasts in ovariectomized postmenopausal mice44. Sorbitol; a
sugar alcohol with a sweet taste has been observed to retard bone resorption in Sprague Dawley male rats45. e
positive eects on osteoporosis prevention by fruits including that of Prunus domestica and Prunus salicina may
partly be due to the presence of sorbitol compound in them1. Recent studies suggest that inammation is one of
the key factors that inuence bone turnover, leading to osteoporosis3. erefore, the potent anti-inammatory
activity of β-sitosterol47; a compound present in P. africana stem bark may further explain its anti-osteoporosis
activity. Stearic acid and oleic acid have been reported to inhibit osteoclastogenesis in bone marrow cultures and
RAW264.7 cells46. Chlorogenic acid was observed to improve the bone quality by modifying the bone mineral
density and trabecular microarchitecture in an ovariectomy rat model49. Furthermore, chlorogenic acid was also
observed to promote proliferation of osteoblast precursors and osteoblastic dierentiation in ovariectomized
rats50. In the present study, chlorogenic acid exhibited signicant inhibition of LPS-induced NO production and
secretion of IL-1β. Interleukin-1 is a very powerful stimulator of bone resorption and is well known to inhibit
bone formation51. e signicant inhibition of IL-1β and NO production by chlorogenic acid indicates that it
plays an important role in preventing and treating osteoporosis. In addition, our study showed that chlorogenic
acid had a signicant inhibition of RANKL-induced TRAP activity giving further evidence of its antiosteoporotic
potential.
In light of these results, the inhibition of the TRAP activity by P. africana bark extracts may therefore be due
to these dierent important compounds in it including chlorogenic acid, catechin, naringenin and β-sitosterol.
ese ndings therefore provide valuable insight in to the anti-osteoporosis potential of P. africana.
Excess production of NO in the body system plays a vital part in pathogenesis of inammatory diseases
including osteoporosis21. It has been reported previously that increased production of NO is a contributing fac-
tor to osteoporosis pathogenesis21,52. us, a potential therapeutic pathway for managing the disease is through
suppression of NO production as indicated by Komakech, etal.21. In this study, the P. africana methanolic
extract (each 150µg/ml) signicantly (p < 0.0001) inhibited NO production actually more than the positive
standard, dexamethasone (40µM). e chemical compounds, chlorogenic acid, catechin, naringenin and
β-sitosterol (100µM each) in RAW264.7 cells also signicantly suppressed LPS-induced NO production. Stud-
ies have reported that high NO concentrations have great inhibitory eects on growth and dierentiation of
osteoblasts36,53–55. is has been suggested to be partly as a result of NO pro-apoptotic eects on osteoblasts56.
e signicant inhibition of LPS-induced NO production by PA extracts and its chemical constituents clearly
demonstrates its antiosteoporotic potential by eliminating the inhibitory eects of NO to growth and dierentia-
tion of osteoblasts. Chlorogenic acid, catehin, naringenin and β-sitosterol did not show toxic eects.
In our current study, PA-methanolic extract (150µg/ml) and β-sitosterol (100µM) just like the positive con-
trol (dexamethasone [40µM and 80µM]) in RAW264.7 cells were observed to signicantly inhibit LPS-induced
IL-6 secretion. is therefore suggests that methanol extracted phytochemical compounds from P. africana stem
bark including β-sitosterol exhibited antiosteoporotic potential by inhibiting the production of IL-6 within the
bone microenvironment. IL-6 is known to potently activate osteoclasts and is responsible for bone resorption34.
Pro-inammatory cytokines notably IL-6 are vital in normal processes of bone remodeling and pathogenesis of
osteoporosis in elderly persons and during perimenopause34. Production of IL-6 induces eminent lytic lesions
along with diuse osteoporosis typical of the disease34. Before menopause, estrogen in bone marrow regulates the
expression of most notably IL-634. IL-6 levels are known to increase with age in not only humans but monkeys
and mice and in the trend with osteoporosis34.
Liver toxicity from herbal and dietary supplements is a common phenomenon and is a leading cause of a num-
ber of underlying liver diseases57. Osteoporosis is a frequent complication in patients with liver complications58.
Indeed, decreased trophic factors such as insulin growth factor in the liver due to liver toxicity or chronic dis-
eases including diabetes my result in osteoblast dysfunction19. erefore, ensuring a healthy liver is fundamental
in maintaining a balanced body biological processes including prevention of bone loss. Zebrash larvae is an
important model system for the evaluation of the liver toxicity when an organism is exposed to a toxicant59.
Liver organogenesis in zebrash initiates at 30 hpf on the le-hand side of the embryo and an enlarged liver bud
connects with the intestine and functionally matures until 72 hpf60. Treatment of the zebrash larvae with liver
toxicants such as tamoxifen reduces liver transparency due to the liver cell death61. In this study, 200µg/ml water
extract and 25µg/ml methanolic extract exhibited maximum concentrations for the acute toxicity. us, tests at
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100µg/ml water extract and 25µg/ml ethanolic extract was used to determine hepatotoxicity in zebrash larvae.
And although each experimental group showed larval mortality, the P. africana extracts did not induce hepato-
toxicity because living larvae did not show liver-specic cell death. Considering that the extraction method can
inuence the composition of extracts62, this may explain dierence in the mortality rate of the zebrash larvae
in the methanolic and water extracts of the P. africana stem bark. Previous studies also showed that P. africana
bark extract administered at 1000mg/kg body weight had no visible deleterious eects on BALB/c mice63 and
showed mild hepatotoxicity and nephrotoxicity in Sprague–Dawley rats64. ese observations therefore showed
that the stem bark of P. africana its non-toxic within a low dose range.
Conclusions
e macrophage-dependent anti-osteoporosis activity of P. africana bark may be attributed to the synergistic
action of the various phytochemicals in its stem bark including chlorogenic acid, catehin, naringenin, vanillic
acid, sorbitol, octadecanoic acid (stearic acid), and β-sitosterol. NO production was signicantly inhibited (all
p < 0.0001) by P. africana methanolic, chlorogenic acid, catehin, naringenin, and β-sitosterol. IL-6 secretion was
signicantly inhibited by P. africana methanolic extract (p < 0.0001) and β-sitosterol (p < 0.0001) and in addi-
tion, chlorogenic acid and naringenin remarkably inhibited IL-1β production. All samples displayed signicant
inhibition of RANKL-induced TRAP activity. Although the methanolic extract of P. africana bark exhibited
potent anti-osteoporosis activity, we recommend that future studies should carry out isolation of the individual
chemicals or group of chemicals that are/is responsible for its anti-osteoporosis activity. Nonetheless, this study
has demonstrated that P. africana bark extracts have no overt hepatotoxic eects in zebrash larvae at a given
dose range and oers a basis for future studies and medicine development with anti-osteoporosis therapeutic
application.
Data availability
e data for this current study are available from the corresponding author upon reasonable request.
Received: 1 June 2021; Accepted: 24 March 2022
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Acknowledgements
is research was funded by the framework of International Cooperation Program (Korea-South Africa Coop-
erative Research Project for Excavation of Candidate Resources of Complementary and Alternative Medicine)
managed by National Research Foundation of Korea (Grant no. 2017093655 and KIOM: D17470). Additionally,
this work was also supported by Development of Foundational Techniques for the Domestic Production of Herbal
Medicines (K18405), Development of Sustainable Application for Standard Herbal Resources (KSN2012320),
Korea Institute of Oriental Medicine through the Ministry of Science and ICT, Republic of Korea. Partially, this
work was also supported by a grant from the Ministry of Trade, Industry and Energy, Republic of Korea (Grant
no. 2019-10063396).
Author contributions
R.K. conceived the original research plans, collected test materials, and wrote this manuscript. K.S. conducted
the experiments on TRAP assays. N.H.Y. and J.L. conducted the extractions of the samples and carried out the
chemical proling. S.K.Y., J.H.S., and G.C. carried out the botanical authentication of the P.africana used in this
study and wrote the manuscript. Y.G.K. performed the statistical analysis and wrote this manuscript. F.O., A.M.,
and G.N.K. collected the sample, wrote, and revised the manuscript. D.O. wrote and revised the manuscript. H.K.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

12
Vol:.(1234567890)
Scientic Reports | (2022) 12:7044 | https://doi.org/10.1038/s41598-022-10629-7
www.nature.com/scientificreports/
and K.S.H. conducted the hepatotoxicity assay on zebrash. M.G.M. conducted experiments on phytochemical
proling and revised the manuscript. Y.K. technically supervised all the experiments and is the corresponding
author. All authors reviewed the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 022- 10629-7.
Correspondence and requests for materials should be addressed to Y.K.
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