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The cancer burden in Africa

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Cancer is currently responsible for more than 7 million deaths
per year worldwide, more than malaria, tuberculosis and
HIV/AIDS combined. There are more than 600 000 deaths
annually in Africa from cancer. In the developing world, the
number of new cancer cases will increase significantly over the
next 10 years. By 2020 there are expected to be 15 million new
cases of cancer every year, 70% of which will be in developing
countries, where governments are least prepared to address the
growing cancer burden and where survival rates are often less
than half those in more developed countries. African countries
will account for over a million new cancer cases a year and
they are the least able of all developing countries to cope,
having fewest cancer care services.
Currently the world is focused on controlling the spread
of HIV, TB and malaria, which are all acknowledged to be
major killers in the developing world. Huge sums of money
are currently available to help combat these diseases. Cancer
is set to become the newest epidemic in the developing world,
claiming a vast number of lives, and there is currently limited
funding available to tackle this disease. Raising awareness
of this looming epidemic in Africa is the first step. If we take
concerted action now, we can prevent another tragedy.
A meeting on Cancer Control in Africa was held on 10 - 11
May 2007 in London to raise awareness of the growing cancer
epidemic in Africa and to determine how best to deliver
comprehensive cancer care to Africa. A new organisation
(AfrOx) set up by Professor D Kerr, Oxford University,
organised the meeting.
Over 130 delegates attended the meeting, which was held
in London’s Reform Club. Twenty African countries (Benin,
Botswana, Burkina Faso, Cameroon, Cape Verde, Egypt, Gabon,
The Gambia, Ghana, Lesotho, Libya, Malawi, Mauritius,
Morocco, Mozambique, Nigeria, Rwanda, Sierra Leone, South
Africa and Zambia) and the Yemen were represented at the
meeting by their Ministers of Health, their representatives, or
their leading oncologists. In addition, representatives from
major national and international health care and cancer-related
charities and organisations including representatives of the
pharmaceutical industry, the World Health Organization, the
World Bank, the International Agency for Research on Cancer
(IARC) and the African Development Bank, members of the
UK parliament, African doctors and health care workers
attended the meeting, together with leading oncologists from
the RSA, UK, USA, France, Netherlands, Ireland, Sweden,
Norway and India.
The aims of the meeting were to: (i) determine the degree of
priority cancer is afforded in national programmes in Africa;
(ii) determine the most affordable and effective components of
cancer control; (iii) decide on a clear implementation strategy
for bringing these programmes to African countries; (iv) design
mentorship and training programmes for African health care
workers and scientists, and engage the support of Oxford
University and the international cancer care community to
run these programmes; and (v) identify a strategy to raise the
necessary funds to enable implementation of the cancer control
programmes.
The African health ministers and their representatives
who presented at the meeting stated unanimously that
they recognise the explosion in cancer incidence and would
welcome the support of the international oncology community
in tackling the growing cancer epidemic, but that in order to
deliver comprehensive cancer control to Africa effectively we
must integrate with existing programmes that are tackling
AIDS, malaria and TB.
This is the first collective and definitive statement by a
representative cross-section of African health ministries of the
urgent need to initiate cancer control programmes. It lays to
rest the myth that the only health priorities in Africa are those
related to infectious diseases and that care of chronic diseases
is best integrated with existing programmes.
In Africa, it is thought that up to a third of cancer deaths
are potentially preventable. In 2002 in sub-Saharan Africa,
there were more than half a million cancer deaths, of which
almost 40% can be explained by chronic infection and tobacco
usage. Chronic infection with the hepatitis virus increases the
risk of liver cancer, infection with HIV increases the risk of
Kaposi’s sarcoma, and chronic infection with certain types of
human papillomavirus increases the risk of cervical cancer.
Today we have vaccines to protect against hepatitis B and
human papillomavirus infection, but they are not available
in the countries that need them most. Tobacco use is the most
preventable cause of death. Unless we see concerted action to
establish cancer prevention programmes (vaccination, anti-
smoking measures, etc.) to reduce the number of cancer cases,
the limited treatment facilities that exist in the majority of
African countries will be completely overwhelmed by an ever-
expanding cancer burden.
Lack of resources and basic infrastructure mean that
most Africans have no access to cancer screening, early
diagnosis, treatment or palliative care. While there have been
some improvements in recent years (in 1991 there were 63
radiotherapy machines and currently there are over 200),
radiotherapy is available in only 21 of Africa’s 53 countries,
reaching less than 5% of the population, and consequently
patients are denied a treatment that can be life-saving and
significantly improve cancer pain.
Some of the startling findings at the London meeting
included: (i) cancer sufferers, when diagnosed, face
The cancer burden in Africa
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stigmatisation in many African countries; (ii) a diagnosis of
cancer is equated with an inescapable and painful death; and
(iii) the majority of attending nations have no elements of a
cancer plan.
The main output from the meeting was the London
Declaration on Cancer Control in Africa (see www.afrox.org),
a document that aims to raise awareness of the magnitude of
the cancer burden in Africa and to call for immediate action to
bring comprehensive cancer care to African countries.
It builds on the World Health Assembly Resolution on
Cancer Prevention and Control (2005) and on previous
declarations from the International Atomic Energy Agency
(Cape Town Declaration on Cancer Control in Africa,
December 2006) and the International Union Against Cancer
(World Cancer Declaration, July 2006).
There was complete agreement among delegates that
the only way effectively to prevent, detect and treat the
rising numbers of cancers in Africa is to develop broad
partnerships between local health care delivery systems,
research institutions, international organisations, national
governments in developed and developing countries, and
the pharmaceutical industry. Strong local and international
leadership is essential. The relevant organisations and
individuals, with funds from government and private
donors, must be brought together to develop achievable and
sustainable national cancer plans that are evidence based,
priority driven and resource appropriate for African countries.
Delegates also agreed that the introduction of cancer care
into African countries requires integration of clinical and
public health systems so that they become truly comprehensive
and bring together prevention, early diagnosis, treatment,
palliative care and the investment needed to deliver these
services in terms of trained staff, equipment, relevant drugs
and information systems. However, any cancer control strategy
must be guided by the needs of the country and must be
resource appropriate for that country.
We believe that we have a timely opportunity to develop
a sustainable model for developing comprehensive cancer
care to African countries, authored by the member states and
with technical, policy and financial support provided by inter-
agency alliances and governments in the developed world.
Daniel A Vorobiof
Sandton Oncology Centre
Johannesburg
Raymond Abratt
Groote Schuur Hospital and University of Cape Town
Cape Town
Corresponding author: D A Vorobiof (voro@tiscali.co.za, voro@global.co.za)
Key reading
Editorial. Cancer treatment: not just a question of costs. Lancet 2007; 369: 1665.
Global Action Against Cancer. Geneva: World Health Organization and International Union against
Cancer, 2005: 1-24.
Kmietowicz Z. Tackle cancer in Africa now to prevent catastrophe, say health activists. BMJ 2007;
334: 1022-1023.
National Institute for Health and Clinical Excellence. Referral Guidelines for Suspect Cancer. London:
NICE, 2005. www.nice.org.uk/pdf/cg027niceguideline.pdf (last accessed 4 September 2007).
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The global cancer burden remains a serious concern with the alarming incidence of one in eight men and one in eleven women dying in developing countries. This situation is aggravated by the multidrug resistance (MDR) of cancer cells that hampers chemotherapy. In this study, the cytotoxicity of the methanol extract (HRB), fractions (HRBa, HRBb, and HRBa1-5), and compounds from the bark of Hypericum roeperianum (HRB) was evaluated towards a panel of 9 cancer cell lines. The mode of action of the HRB and trichadonic acid (1) was also studied. Column chromatography was applied to isolate the constituents of HRB. The cytotoxicity of botanicals and phytochemicals was evaluated by the resazurin reduction assay (RRA). Caspase-Glo assay was used to evaluate the activity of caspases, and reactive oxygen species (ROS) (H2DCFH-DA) were assessed by flow cytometry. Phytochemicals isolated from HRB were trichadonic acid (1), fridelan-3-one (2), 2-hydroxy-5-methoxyxanthone (3), norathyriol (4), 1,3,5,6-tetrahydroxyxanthone (5), betulinic acid (6), 3′-hydroxymethyl-2′-(4″-hydroxy-3″,5″-dimethoxyphenyl)-5′,6′:5,6-(6,8-dihydroxyxanthone)-1′,4′-dioxane (7), and 3′-hydroxymethyl-2′-(4″-hydroxy-3″,5″-dimethoxyphenyl)-5′,6′:5,6-(xanthone)-1′,4′-dioxane (8). Botanicals HRB, HRBa, HRBa2-4, HRBb, and doxorubicin displayed cytotoxic effects towards the 9 tested cancer cell lines. The recorded IC50 values ranged from 11.43 µg/mL (against the P-glycoprotein (gp)-overexpressing CEM/ADR5000 leukemia cells) to 26.75 µg/mL (against HCT116 (p53+/+) colon adenocarcinoma cells) for the crude extract HRB. Compounds 1, 5, and doxorubicin displayed cytotoxic effects towards the 9 tested cancer cell lines with IC50 values varying from 14.44 µM (against CCRF-CEM leukemia cells) to 44.20 µM (against the resistant HCT116 (p53−/−) cells) for 1 and from 38.46 µM (against CEM/ADR5000 cells) to 112.27 µM (against the resistant HCT116 (p53−/−) cells) for 5. HRB and compound 1 induced apoptosis in CCRF-CEM cells. The apoptotic process was mediated by enhanced ROS production for HRB or via caspases activation and enhanced ROS production for compound 1. This study demonstrated that Hypericum roeperianum is a potential source of cytotoxic phytochemicals such as trichadonic acid and could be further exploited in cancer chemotherapy. 1. Introduction Cancer continues to be a global threat, appearing as the second leading cause of death globally, with estimated 9.6 million deaths representing one in six deaths and with an estimated five-year prevalence of 43.8 million people [1]. Multidrug resistance (MDR) of cancer cells is a serious concern in chemotherapy. It is responsible for many therapeutic failures and high burdens globally, in patients suffering from cancer [2, 3]. Any modern protocol for new cytotoxic drug discovery today should integrate the ability of neoplastic cells to rapidly develop resistant phenotypes. Thus, resistant cell lines should be integrated into the cell panel used for the discovery of more efficient substances. The present work has taken this into account and involves several models of MDR cancer cell lines such as the colon adenocarcinoma with p53 knockout phenotype, the leukemia cells with ATP-binding cassette (ABC)-transporter-overexpressing MDR-mediating-P-glycoprotein (P-gp; ABCB1/MDR1), the breast cancer bearing resistance protein (ABCG2/BCRP), and the transfectant glioblastoma multiforme harboring a mutation-activated EGFR gene (ΔEGFR). The effectiveness of natural products in the fight against cancer has been largely demonstrated [4]. Some clinically established cytotoxic drugs such as camptothecin, paclitaxel, vinblastine, or vincristine are naturally occurring compounds [4–6]. In addition, numerous botanicals and phytochemicals derived from African medicinal plants have been found active against MDR cancer cell lines [7, 8]. Some of such prominent phytochemicals include terpenoids: salvimulticanol and candesalvone B methyl ester [9], epunctanone [10], and ardisiacrispin B [11], phenolics: 2-acetyl-7-methoxynaphtho [2,3-b]furan-4,9-quinone [12], 6α-hydroxyphaseollidin [13], licoagrochalcone A [14], 7-dihydroxy-4′-methoxy-6,8-diprenylisoflavone, and 7,7″-di-O-methylchamaejasmin [15], and alkaloids: 1,3-dimethoxy-10-methylacridone [16], isotetrandrine [17], and ungeremine [18]. However, more hit compounds should be identified to increase our arsenal of cytotoxic compounds and to secure better the chances of later obtaining new clinically usable molecules. The present study was, therefore, designed to assess the cytotoxicity of botanicals and phytochemicals from the bark of Hypericum roeperianum Schimp. p. ex A. Rich (Guttiferae). The modes of action of compound 1, such as its effects on cell cycle distribution and induction of apoptosis, on caspases activation, and on the production of reactive oxygen species (ROS), were also investigated. Hypericum roeperianum is a shrub or small tree growing in the tropical part of central, eastern, and southern tropical Africa, locally used alone or in association with various plants in the treatment of female sterility [19], as antiabortifacients [20] and as antifungal remedies [21]. Previous phytochemical investigations of this plant led to the isolation of a polyketide, 4-methoxy-3-(2-methylbut-3-en-2-yl)-6-phenyl-2H-pyran-2-one, xanthones: 1,5-dihydroxy-6-methoxyxanthone, 2-hydroxy-5-methoxyxanthone and 1,4,6,7-tetrahydroxyxanthone, and the xanthonolignoids: 8,10-dihydroxy-3-(4hydroxy-3,5-dimethoxyphenyl-2-(hydroxymethyl)-2, 3-dihydro-[1, 4]dioxino[2,3-c]xanthen-7-one and 8-hydroxy-10-methoxy-3-(4-hydroxy-3,5-dimethoxyphenyl-2-(hydroxymethyl)-2,3-dihydro-[1,4]dioxino[2,3-c]xanthen-7-one from the bark [22] and 10 other xanthones from the roots, namely, 5-O-methyl-2-deprenylrheediaxanthone B, 5-O-methylisojacareubin, 5-O-demethylpaxanthonin, roeperanone, 2-hydroxyxanthone, 5-hydroxy-2-methoxyxanthone, 1,5-dihydroxy-2-methoxyxanthone, 2-deprenyl rheediaxanthone B, isojacareubin, and calycinoxanthone D [23]. The cytotoxicity of botanicals from the bark of Hypericum roeperianum is being reported for the first time. 2. Material and Methods 2.1. Chemicals Doxorubicin (98.0% purity) from Sigma-Aldrich (Munich, Germany) was obtained from the Johannes Gutenberg University Medical Center (Mainz, Germany). Geneticin >98% (used at 800 ng/mL and 400 µg/mL) in culture media to maintain the features of MDA-MB-231-BCRP, U87MG.ΔEGFR, and HCT116 (p53⁻/⁻), respectively, was obtained from Sigma-Aldrich and stored at 72.18 mM. Hydrogen peroxide (H2O2) and valinomycin (at 1 mg/mL) were provided by Sigma-Aldrich (Taufkirchen, Germany). 2.2. Plant Material and Extraction The bark of Hypericum roeperianum Schimp. p. ex A. Rich (Guttiferae) was collected in Bangang Wabane (South West Region of Cameroon) in October 2018. No permission was necessary for sample’s collection. The identification of the plant was carried out by Dr. Tchiengue Barthelemy at the Cameroon National Herbarium (Yaoundé) where a voucher specimen was deposited under the number 24584/SRF/Cam. Dried barks of the plant (3.0 kg) were powdered and extracted with methanol (MeOH; 3 × 15 L) for 72 h at room temperature to afford a crude extract (HRB; 150.0 g) after filtration with Whatman paper no.1 and evaporation in vacuum, under reduced pressure. A portion of the resulting extract (140.0 g) was, then, exhausted in ethyl acetate to yield 65.0 g of the ethyl acetate extract (EtOAc) (HRBa) and the residue (HRBb; 75 g). 2.3. Fractionation and Purification of the Bark Extract of Hypericum roeperianum Part of the ethyl acetate extract (EtOAc; 60.0 g) was submitted to silica gel flash chromatography using hexane-EtOAc and, then, EtOAc-MeOH mixtures of increasing polarities. Eighty fractions (frs) of 500 mL each were collected as follows: hexane 100% (sub-frs 1–3), hexane-EtOAc 90 : 10 (sub-frs 4–12), hexane-EtOAc 80 : 20 (sub-frs 13–18), hexane-EtOAc 70 : 30 (sub-frs 19–22), hexane-EtOAc 60 : 40 (sub-frs 23–27), hexane-EtOAc 50 : 50 (sub-frs 28–37), hexane-EtOAc 30 : 70(sub-frs 38–43), AcOEt 100% (sub-frs 44–52), EtOAc-MeOH 95 : 5 (sub-frs 53–57), EtOAc-MeOH 90 : 10 (sub-frs 58–62), EtOAc-MeOH 80 : 20 (sub-frs 63–69), and MeOH 100% (sub-frs 70–80). Based on their analytical thin-layer chromatography (TLC) profiles, these fractions were pooled into five fractions (frs) as follows: HRBa1 (Sub-frs 1–15, 8.0 g), HRBa2 (Sub-frs 16–25, 15.0 g), HRBa3 (Sub-frs 28–38, 9.5 g), HRBa4 (Sub-frs 39–68, 12.5 g), and HRBa5 (Sub-frs 69–80, 13.0 g). Dry fraction HRBa2 (15.0 g) was dissolved in methanol affording a nonsoluble powder which was, then, filtered to give compound 1 (15 mg). The filtrate was subjected to silica gel column chromatography using hexane-AcOEt mixtures of increasing polarities as elution solvents. Sixty-five sub-frs of 150 mL each were collected as follows: Hex 100% (1–3), Hex-AcOEt 90:10 (Sub-frs 4–9), Hex-AcOEt 80 : 20 (Sub-frs 10–15), Hex-AcOEt 70 : 30 (Sub-frs 16–19), Hex-AcOEt 60 : 40 (Sub-frs 20–35), Hex-AcOEt 50 : 50 (Sub-frs 36–42), Hex-AcOEt 40 : 60 (Sub-frs 43–50), AcOEt 100% (Sub-frs 51–55), AcOEt-MeOH 90 : 10 (Sub-frs 56–60), and MeOH 100% (Sub-frs 61–65). Sub-frs 6–9 yielded compound 2 (15.0 mg) as a white powder. Sub-frs EC23-29 yielded compound 3 (12.0 mg) as a yellow powder. HRBa3 (9.5 g) was subjected to silica gel column chromatography using Hex-AcOEt mixtures of increasing polarities as elution solvents. Seventy-five sub-frs of 150 mL each were collected as follows: hexane 100% (sub-frs 1–4), Hex-AcOEt 90 : 10 (sub-frs 5–9), Hex-AcOEt 80 : 20 (sub-frs 9–16), Hex-AcOEt 70 : 30 (sub-frs 17–23), Hex-AcOEt 60 : 40 (sub-frs 24–35), Hex-AcOEt 50 : 50 (sub-frs 36–42), Hex-AcOEt 40 : 60 (sub-frs 43–47), AcOEt 100% (sub-frs 48–57), AcOEt-MeOH 90 : 10 (sub-frs 58–63), AcOEt-MeOH 80 : 20 (sub-frs 64–70), and MeOH 100% (sub-frs 71–75). Sub-frs 22–25 yielded compound 4 (18.0 mg) as a green-yellowish powder. Sub-frs 29–35 yielded compound 5 as a yellow powder (15.0 mg). HRBa4 (12.5 g) was subjected to silica gel column chromatography using CH2Cl2-MeOH mixtures of increasing polarities as elution solvents. Fifty sub-frs of 150 mL each were collected as follows: CH2Cl2 100% (sub-frs 1–4), CH2Cl2-MeOH 95 : 5 (sub-frs 5–13), CH2Cl2-MeOH 90 : 10 (sub-frs 14–25), CH2Cl2-MeOH 85 : 15 (sub-frs 26–35), CH2Cl2-MeOH 80 : 20 (sub-frs 36–42), and MeOH 100% (subfrs 43–50). Sub-frs 6–11 yielded compound 6 (40.0 mg) as a white powder. Sub-frs 13–15 yielded compound 7 (12.0 mg) as a yellow powder. Sub-frs 19–23 yielded compound 8 (14.0 mg) as a yellow powder. 2.4. Cell Cultures Cell lines used in this work included drug-sensitive and drug-resistant phenotypes of earlier reported origin. They were all provided by Prof. Dr. Thomas Efferth from his cell lines collection; they have being used in cytotoxicity screening by our team for a decade [12–21]. These include two hematological cancer cell lines, namely, the drug-sensitive CCRF-CEM leukemia cell line and its multidrug-resistant P-gp-over-expressing subline CEM/ADR5000 cells [24–26] and nine carcinoma cell lines, namely, U87.MG glioblastoma cell line and its EGFR-transfected U87.MGΔEGFR subline, HCT116 (p53+/+) colon cancer cell line and its knockout clone HCT116 (p53⁻/⁻), and MDA-MB-231-pcDNA3 breast cancer cell line and its BCRP-transfected multidrug-resistant MDA-MB-231-BCRP clone 23 cell line [27], as well as the normal AML12 hepatocytes, used to compare with HepG2 liver cancer cells [13]. 2.5. Resazurin Reduction Assay (RRA) for Cell Growth Evaluation The RRA was applied to evaluate the cytotoxicity of botanicals, the isolated phytochemicals (1–5, 7, and 8), and doxorubicin on the cell growth as reported earlier [18, 28]. Cells treated with various samples at different concentrations were incubated for 72 h in humidified 5% CO2 atmosphere at 37°C. Cells were further coloured with resazurin and incubated for 1–2 h; the fluorescence was further measured with an Infinite M2000 Pro™ plate reader (Tecan, Crailsheim, Germany) at 544 nm as the excitation wavelength and 590 nm as the emission wavelength. The IC50 values represented the concentrations of the sample required to inhibit 50% of cell proliferation and were calculated from a calibration curve by linear regression using Microsoft Excel 2007 [29]. 2.6. Flow Cytometric Evaluation of Cell Cycle Distribution and Apoptotic Cells Various concentrations of botanical HRB, phytochemical 1, and doxorubicin or DMSO (solvent control) were used to treat CCRF-CEM cells (1 × 10⁶ cells). Cells were further incubated for 24 h in humidified 5% CO2 atmosphere at 37°C and analyzed using a BD Accury C6 Flow Cytometer (BD Biosciences, Heidelberg, Germany) by measuring the propidium iodide fluorescence of individual nucleus, as described earlier [10, 11]. Experiments were conducted thrice independently with three parallel measurements. 2.7. Assessment of Apoptosis by Annexin V/PI Staining The CCRF-CEM cells (1 × 10⁶; 1 ml) were also treated with HRB, compound 1 and doxorubicin for 24 h (in humidified 5% CO2 atmosphere at 37°C), and apoptosis was further assessed by flow cytometry using the flouresceinisothiocynate- (FITC-) conjugated annexin V/PI assay kit (eBioscience™Annexin V; Invitogen, San Diego, USA), as previously published [10, 11]. Briefly, treated cells were centrifuged at 1200 rpm for 5 min, then washed twice with ice-cold PBS, resuspended in 500 µl binding buffer, and stained with 5 µl FITC-conjugated annexin V (10 mg/mL) and 10 µl PI (50 mg/ml). After 15 min incubation at room temperature (RT) in the dark, cells were analyzed using a BD Accury C6 Flow Cytometer (BD Biosciences). Cells stained with only annexin V were evaluated as being in early apoptosis. Cells stained with both annexin V and propidium iodide were evaluated as being in late apoptosis or in a necrotic stage. 2.8. Evaluation of Caspases Activities Using Caspase-Glo 3/7, Caspase-Glo 8, and Caspase-Glo 9 Different concentrations of HRB and compound 1 were used to treat CCRF-CEM cells for 6 h. The activities of caspases were determined using Caspase-Glo 3/7, Caspase-Glo 8, and Caspase-Glo 9 Assay kits (Promega, Mannheim, Germany) by measuring the luminescence using an Infinite M2000 ProTM plate reader (Tecan), as reported previously [13]. 2.9. Evaluation of Reactive Oxygen Species (ROS) Production Various concentrations of HRB and triterpenoid 1 were used to treat CCRF-CEM cells (1 × 10⁶ cells); DMSO (solvent control); or hydrogen peroxide (H2O2; positive control). After 24 h incubation in humidified 5% CO2 atmosphere at 37°C, the production of ROS was evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFH-DA) (Sigma-Aldrich) staining, as described earlier [30–32]. 2.10. Statistics Statistical analyses were performed with Graph pad prism 5 software. Representative data from three independent experiments are shown as mean value ± S.E.M. One-way Analysis Variance (ANOVA) followed by post hoc Tukey’s test was used to determine the significance of the difference between mean values relative to the control. The value was calculated to determine significant differences ( value < 0.05). 3. Results 3.1. Phytochemistry The chemical structures of the isolated phytochemicals were determined by exploiting the physical, mass spectra, and NMR data, followed by direct comparison of these data with those of similar reported compounds in the literature. Compounds were identified as trichadonic acid C30H48O3 (1; white amorphous powder; m/z 456) [33], fridelan-3-one C30H50O (2; white powder; m.p. 258oC; m/z 426) [33], 2-hydroxy-5-methoxyxanthone C14H10O4 (3; yellow amorphous powder; m/z 242) [34], 1,3,6,7-tetrahydroxyxanthone or norathyriol C13H8O6 (4; green-yellowish powder; m.p. 271oC; m/z 260) [35], 1,3,5,6-tetrahydroxyxanthone C13H8O6 (5; yellow powder; m.p. 136oC; m/z 260) [36], betulenic acid C30H48O3 (6; white powder; m.p. 318oC; m/z 456) [33], 3′-hydroxymethyl-2′-(4″-hydroxy-3″,5″-dimethoxyphenyl)-5′,6′:5,6-(6,8-dihydroxyxanthone)-1′,4′-dioxane C24H20O8 (7; yellow powder; m.p. 264oC; m/z 436) [37, 38], and 3′-hydroxymethyl-2′-(4″-hydroxy-3″,5″-dimethoxyphenyl)-5′,6′:5,6-(xanthone)-1′,4′-dioxane C2420 O10 (8; yellow amorphous powder; m/z 468) [37] (Figure 1). The 1D NMR spectra of these compounds are provided as Supplementary Materials.
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