|Year : 2017 | Volume
| Issue : 2 | Page : 168-173
Potential roles of Kleinhovia hospita L. leaf extract in reducing doxorubicin acute hepatic, cardiac and renal toxicities in rats
Yulia Yusrini Djabir1, M Aryadi Arsyad2, Sartini Sartini3, Subehan Lallo4
1 Laboratory of Clinical Pharmacy, Faculty of Pharmacy, Hasanuddin University, Makassar, Indonesia
2 Department of Physiology, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia
3 Laboratory of Microbiology, Faculty of Pharmacy, Hasanuddin University, Makassar, Indonesia
4 Laboratory Phytochemistry, Faculty of Pharmacy, Hasanuddin University, Makassar, Indonesia
|Date of Web Publication||18-Apr-2017|
Yulia Yusrini Djabir
Laboratory of Clinical Pharmacy, Faculty of Pharmacy, Hasanuddin University, Jl. Perintis Kemerdekaan, KM.10, Tamalanrea 90245, Makassar
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Doxorubicin (DOX) is a potent chemotherapy agent; however, its use may lead to cardiac, hepatic, and renal dysfunction. Kleinhovia hospita L extract contains antioxidant compounds that have been shown to reduce chemical-induced hepatotoxicity. Objectives: This study aimed to examine the protective effects of Kleinhovia sp. extract to reduce DOX acute toxicities. Materials and Methods: Thirty male rats were assigned to the following groups: Group I as controls, Group II was given DOX i.p. injection (25 mg/kg); Groups III, IV, and V were treated with Kleinhovia sp. extract 100, 250, and 500 mg/kg orally for 5 days, respectively, prior to DOX i.p. injection. After 24 h, blood and organs were analyzed for biomarker levels and histopathological changes. Results: DOX treatment in Group II significantly increased creatine kinase-MB (CK-MB), aspartate transaminase (AST), alanine transaminase (ALT), and urea levels compared to controls. Kleinhovia sp. extract at any given dose significantly improved ALT and AST; yet, CK-MB levels only reduced with 250 mg/kg dose (Group IV). Urea and creatinine levels in Kleinhovia sp. groups were also lower compared to DOX-treated rats, but it was not significant. Histopathological analysis showed improved liver, heart, and renal tissue structures in Kleinhovia sp-treated rats, especially at higher doses. Conclusion: Kleinhovia sp. extract at any dose given protected the rats from liver toxicity, but only at dose 250 mg/kg reduced cardiac toxicity. Although renal biomarkers were insignificantly lower, renal architecture was improved with Kleinhovia sp. treatment.
Keywords: Acute toxicity, doxorubicin, Kleinhovia sp. extract
|How to cite this article:|
Djabir YY, Arsyad M A, Sartini S, Lallo S. Potential roles of Kleinhovia hospita L. leaf extract in reducing doxorubicin acute hepatic, cardiac and renal toxicities in rats. Phcog Res 2017;9:168-73
|How to cite this URL:|
Djabir YY, Arsyad M A, Sartini S, Lallo S. Potential roles of Kleinhovia hospita L. leaf extract in reducing doxorubicin acute hepatic, cardiac and renal toxicities in rats. Phcog Res [serial online] 2017 [cited 2020 Feb 25];9:168-73. Available from: http://www.phcogres.com/text.asp?2017/9/2/168/204646
- Doxorubicin (25 mg/kg) i.p injection led to elevated ALT, AST, CK-MB and urea levels in rats.
- At the given dose, doxorubicin induced pathological changes in cardiac, liver and renal tissues.
- Pretreatment with Kleinhovia sp. extract prior to doxorubicin injection significantly reduced the elevation of ALT, AST and CK-MB, especially at the dose of 250 mg/kg.
- Improvement in histological structures of cardiac, liver and renal tissues was shown in Kleinhovia sp. (250 mg/kg) treated rats, indicating a protective effect of the extract on doxorubicin acute toxicity.
Abbreviations Used: DOX: Doxorubicin; CK-MB: creatine kinase-MB, AST: Aspartate transaminase; ALT: Alanine transaminase
| Introduction|| |
Doxorubicin (DOX) is a potent chemotherapy and widely used for many cancer types. However, it may lead to cardiac toxicity when used chronically or in a high dose, leading to myocardial dysfunction in cancer patients. Recently, DOX is also found to elicit hepatic and renal toxicity,, raising more concern for DOX chemotherapy regardless its potential effects against cancer cells.
Kleinhovia hospita L is known as Paliasa or Tahongai in Indonesia, and its leaves have been empirically used to treat hepatitis.Kleinhovia sp. hepatoprotection is mostly derived from its natural compounds, cycloartane triterpenoid alkaloids (Kleinhospitines A-D), which are shown to protect liver cells from H2O2 oxidative stress., Moreover, Kleinhovia sp. leaves also contain kaempferol 3-O-β-glucoside and eleuterol, which act as antioxidants. Indeed, hepatoprotective effects of Kleinhovia sp. have been demonstrated against paracetamol-induced hepatotoxicity., It is hypothesized that antioxidant compounds in Kleinhovia sp. leaves may also protect cells from DOX toxicity, especially in the liver and possibly in the heart and kidney. Therefore, the aim of this study is to examine the effect of Kleinhovia sp. extract administration prior to i.p. injection of DOX on liver, cardiac, and renal acute toxicities.
| Materials and Methods|| |
Materials and extracts
DOX for injection (50 mg/25 ml) was purchased from Kalbe Farma (Makassar, Indonesia). Fresh Kleinhovia sp. leaves were obtained from Makassar, Indonesia, and dried in shade before cut into small pieces. Dried leaves were extracted with maceration in 70% ethanol for 3 days. Extract was thickened using a rotary evaporator and then prepared in three different concentrations (1%, 2.5%, and 5%) suspended in natrium carboxy-methyl-cellulose (NaCMC) 1%. Extract suspension was administered accordingly to obtain the doses of 100, 250, and 500 mg/kg body weight in rats. The dose of Kleinhovia sp. extract used in this study was selected based on a previous study showing hepatoprotective effects of Kleinhovia sp.
Male Wistar rats 200–250 g (n = 30) were caged with food and water ad libitum. Animals were cared for in accordance with the institutional standard for using experimental animals. Animals were adapted in the cage for 2 weeks before conducting experiment. The project was registered with ethical number of UH16101030.
Experimental protocol is depicted in [Figure 1]. Rats were assigned into one of the five groups. Blood samples were withdrawn 1 day before any treatment given to obtain baseline data. Group I was assigned as control rats and only given NaCMC 1% suspension for 5 days and then injected with NaCl 0.9% on the 5th day. Group II was given NaCMC 1% suspension for 5 days and a single shot of DOX (i.p. 25 mg/kg) on the 5th day. Groups III, IV, and V were, respectively, given Kleinhovia sp. extract orally with the dose of 100 mg/kg, 250 mg/kg, and 500 mg/kg for 5 days prior to DOX i.p. injection (day 5). After 24 h from DOX injection, rats were anesthetized with diethyl ether, and 3 ml of blood samples was obtained from rats' lateral tail veins using BD-Vacutainer® tube with ethylenediaminetetraacetic acid. Blood samples were centrifuged (Hettich®) at a speed of 2000 rpm for 25 min. The plasma was removed immediately and stored in −20°C until biomarker analysis was performed.
|Figure 1: Diagram of experimental protocols. Animals were adapted for 14 days prior to Kleinhovia sp. extract administration for 5 consecutive days. At day 5, rats were injected with either saline or doxorubicin. After 24 h, blood was withdrawn and organs were harvested for further analysis|
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Plasma biomarker creatine kinase-MB (CK-MB), aspartate transaminase (AST), alanine transaminase (ALT), urea, and creatinine were measured according to the diagnostic kit instructions specific for Humalyzer 3500 (Human®) instrumentation.
After withdrawing the blood, rats were euthanized with cervical dislocation. Heart, liver, and kidneys were carefully removed, fixated in 10% buffered formaldehyde, and embedded in paraffin. Sections of 5 μm thick were serially sliced with a microtome and stained with hematoxylin and eosin. Two subsequent sections of each organ sample were examined by an investigator blinded to the treatment groups using a microscope and photographed with a digital sight camera (Nikon Eclipse 50i).
Results are reported as mean ± standard error of mean for each group. Normal distribution was analyzed with 1-K sample Kolmogorov–Smirnov test. Normally distributed data were analyzed with one-way ANOVA followed by Tukey's honest significant difference post hoc test. P < 0.05 was considered statistically significant.
| Results|| |
All biomarker levels measured before treatments showed a normal range for rat plasma biomarkers and shown in [Table 1].
Two parameters were used to indicate liver toxicity: ALT and AST. ALT is more sensitive and specific as an indicator of liver cell integrity and function, thus increased level of ALT is an excellent predictor to indicate liver damage. Meanwhile, AST is found equally abundant in the liver and cardiac cells, hence AST elevation may detect either liver or cardiac damage or both. The result shows that ALT levels in Group II significantly elevated 24 h after treated with DOX only. In contrast, rats treated with Kleinhovia sp. extract 5 days before i.p. injection of DOX did not experience ALT elevation, and this was shown in all Kleinhovia sp. groups independent of the dose given [Figure 2].
|Figure 2: Levels of alanine transaminase, aspartate transaminase, creatine kinase-MB, urea, and creatinine in rats treated with doxorubicin only and Kleinhovia sp. Ethanolic extract. Data expressed in mean ± standard error of mean. Group I: controls, Group II: doxorubicin only, Group III: Kleinhovia sp. 100 mg/kg + doxorubicin; Group IV: Kleinhovia sp. 250 mg/kg + doxorubicin; Group V: Kleinhovia sp. 500 mg/kg + doxorubicin. *P<0.05 compared with doxorubicin group (group II)|
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DOX-treated rats also had increased level of AST, twice as much as the normal controls (P < 0.05). Interestingly, AST elevation was also evident in groups that received Kleinhovia sp. extract at doses 100 and 500 mg/kg (Group III and V) in spite of a significant reduction in ALT levels in those groups. It is believed that the increased AST level was dominantly released from damaged cardiac cells rather than liver cells as the ALT levels in those groups were near normal. In contrast, the rats treated with 250 mg/kg of Kleinhovia sp. extract had significantly lower AST level compared to DOX-treated rats [Figure 2]. This result indicates that Kleinhovia sp. extract was protective in hepatic cells from the dose of 100 to 500 mg/kg, but it was only protective in cardiac cells with the dose of 250 mg/kg.
This hypothesis is also supported by the result of CK-MB analysis, which is a sensitive and specific biomarker for myocardial infarction. It is shown that CK-MB level increased almost 4 folds after DOX injection [Figure 2]. With the doses of 100 and 250 mg/kg, Kleinhovia sp. extract was effective to reduce CK-MB level, but apparently, dose 250 mg/kg provided more protection. In contrast, the high dose of Kleinhovia sp. extract (500 mg/kg) failed to reduce cardiac biomarkers, both AST and CK-MB, and even led to an elevation of CK-MB marker in two out of six animals in the group.
For renal toxicity evaluation, two biomarkers were measured: urea and creatinine. Creatinine is a more specific marker to depict renal function compared to urea, but both markers are widely used in clinical setting to monitor renal function. The data show that DOX injection led to significantly increased urea level but did not markedly increase creatinine level. Administration of Kleinhovia sp. extract appears to comparatively lower the urea levels after DOX injection (III, IV, V) even though it is not considered statistically significant [Figure 2]. Although DOX-treated animals did not show a significant elevation of plasma creatinine, there was a trend of lower creatinine level in Kleinhovia sp-treated Groups (III and V) compared to Group II.
In liver tissues [Figure 3], control rats showed normal architecture of hepatocytes. However, after 24 h of DOX i.p. injection (Group II), rat livers showed extensive degeneration of hepatocytes, hydropic changes, with signs of necrotic cells and vacuoles. Kleinhovia sp. treatments prior to DOX injection led to improved structures, which was dose dependent. In a lower dose (100 mg/kg), rat livers were shown to have some necrotic cells and vacuoles but it was not as extensive as Group II. Using higher doses (Group IV and V), administration of Kleinhovia sp. extract led to a significant improvement of tissue architecture although signs of congestion were still apparent in both groups.
|Figure 3: Representative of liver tissues in controls, rats treated with doxorubicin only or with Kleinhovia sp. controls (a) had normal liver structure, Group II (b) shows extensive damage with necrotic cells (black arrow), degeneration and vacuoles (green arrow). Group III with Kleinhovia sp. 100 mg/kg (c) shows slightly damaged area, necrotic cells, and vacuoles. Group IV with Kleinhovia sp. 250 mg/kg (d) shows improved structures with congested vessel (black arrow). Group V with Kleinhovia sp. 500 mg/kg (e) had congested vessels but hepatic tissue mostly improved|
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In cardiac tissue, treatment with DOX only (Group II) caused significant structural changes in myocardium of rats, including loss of cross striation and eosinophilic cytoplasm, which indicate progression of myocardial infarction. Scattered pyknotic nuclei were also evident in myocardium of rats in Group II. Eosinophilic area and loss of cross striation were still apparent in rats treated with Kleinhovia sp. extract 100 mg/kg (Group III), as well as in rats treated with 500 mg/kg (Group V) in a less rigorous manner. Meanwhile, treatment with 250 mg/kg Kleinhovia sp. extract (Group IV) showed significantly improved myocardial structure [Figure 4].
|Figure 4: Representative of myocardial tissue in controls, rats treated with doxorubicin only or with Kleinhovia sp. Group control (a) shows a normal myocardial architecture. Group II with doxorubicin only (b) had loss of cross striation, pyknotic cell (black arrow), and congestion (red arrow). Group III with Kleinhovia sp. 100 mg/kg (c) shows hemorrhage and pyknotic cells and loss of cross striation. Group IV with Kleinhovia sp. 250 mg/kg (d) shows near-normal myocardium. Group V with Kleinhovia sp. 500 mg/kg (e) shows absence of cross striation and hemorrhage (red arrow)|
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In renal tissue, control rats showed a normal architecture of glomerulus and tubules. In rats treated with DOX only, the structure of renal tubules was significantly altered, which mostly showed swelling, hydropic degeneration of tubules, and vacuolization. Necrotic changes and pyknotic cells were also evident in Group II. In Kleinhovia sp-treated rats with dose of 100 mg/kg (Group III), the renal tubules still showed hydropic degeneration and vacuolization, with hemorrhagic glomerulus. With higher doses of extract (250 mg/kg and 500 mg/kg), less pathological changes were found in renal tubules, but glomerular hemorrhage is still evident [Figure 5].
|Figure 5: Representative of renal histology in controls, rats treated with doxorubicin only or with Kleinhovia sp. Group I (a) shows normal tubule architecture. Group II (b) shows extensive damage with tubule hydropic degeneration (yellow arrow), vacuolization (green arrow), and necrotic cells (black arrows). Group III with Kleinhovia sp. 100 mg/kg (c) shows hemorrhagic glomerulus with dilated tubules. Group IV with Kleinhovia sp. 250 mg/kg (d) shows improved tubule structure with hemorrhagic glomerulus. Group V with Kleinhovia sp. 500 mg/kg (e) shows near-normal renal tubules with hemorrhage (red arrow)|
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| Discussion|| |
Regardless of its potent chemotherapy action, DOX administration often leads to complications in patients with cancer. Due to its cardiotoxicity, the prevalence of cardiovascular diseases in patients with breast cancer significantly increases with DOX chemotherapy. This necessitates the search of cytoprotective agents that may halt DOX toxicity. Toxic effects of DOX are triggered from its conversion to semiquinone form, which is further converted into free radical metabolites (C7-radical DOX). These metabolites, in turn, increase the formation of reactive oxygen species (ROS), which lead to oxidative stress  and cellular toxicities.
Some animal studies have been conducted to find better protection against DOX toxicity.,,, Although many of them have shown promising results, only iron chelating agent, dexrazoxane, has been clinically approved as an adjuvant therapy to reduce DOX-induced cardiotoxicity so far. This present study shows that Kleinhovia sp leaf extract may also provide benefits to alleviate hepatotoxicity and cardiotoxicity of DOX although its benefit in DOX-induced nephrotoxicity was not as evident.
In this study, a single injection of DOX (25 mg/kg) triggered a significant elevation of the liver and cardiac cell biomarkers, indicating its deleterious effects on those organs' function. Toxic effect of DOX is associated with increased formation of ROS, profuse release of pro-inflammatory cytokines and inflammation, and induction of apoptotic and necrotic changes in these organs., The use of Kleinhovia sp. (especially in dose of 250 mg/kg) prior to DOX injection led to reduced ALT, AST, and CK-MB compared to those of DOX-treated rats [Figure 2], indicating a superior protection toward liver and cardiac cells. Interestingly, analysis for renal biomarkers did not show a significant difference between DOX-only and Kleinhovia sp-pretreated animals.
Histopathological examination reveals that DOX induces extensive damage to the liver, heart, and renal structures. This present study showed that administration of higher doses of Kleinhovia sp. (250 and 500 mg/kg)before DOX injection could maintain hepatocyte structures with few histological changes, including congestion. Congestion might be, in part, a result of anesthetic use during anesthesia prior to euthanasia. In cardiac tissue, a near-normal histological appearance was shown in animals treated with 250 mg/kg Kleinhovia sp. Although Kleinhovia sp. extract might also improve renal tubule integrity [Figure 5], yet the glomerulus still shows signs of hemorrhage in Kleinhovia sp-treated animals at any given dose. This may suggest that protective effect of Kleinhovia sp. against DOX toxicity is limited in renal tissue.
Leaves of Kleinhovia sp. from family of Sterculiaceae are known to contain flavonoids such as kaempferol, quercetin, and rutin. Further studies have isolated a range of bioactive compounds from this plant. These include kaempferol 3-O-b-D-glucoside and eleuthero, cycloartane triterpenoids including gardenolic acid B, and Kleinhospitines A-D. More recently, Mo et al. isolated two new cycloartane triterpenoids form of K. hospita, namely (23R)-21,23:23,27-diepoxycycloarta-1,24-diene-3,27-dione and (3α)-(α-L-arabinopyranosyloxy)-1α-hydroxy-23-oxocycloartan-28-oic acid. Most of these bioactive compounds showed potent antioxidant activities and cytotoxicity against cancerous cells,,, as well as promising hepatoprotective effects. Currently, extracts of K. hospita are mostly used in Indonesian communities for treating hepatitis and hepatotoxicity, but its potential use as an antitumor agent has now gained more attention.,
| Conclusion|| |
Kleinhovia sp. extract has potential to reduce DOX-induced liver and cardiac damage, especially in the dose of 250 mg/kg. This contention is also supported by improved hepatic and cardiac tissue architecture following Kleinhovia sp. extract administration. Regardless of some improvement in renal tubule histology, administration of Kleinhovia sp. ethanolic extract has a limited impact on reducing renal biomarkers.
The authors would like to thank Ismail and Muhammad Nur Amir for their support on sample collection and animal care. We would also acknowledge that part of the data has been presented in the 2nd Indonesian Conference on Clinical Pharmacy in Bali, Indonesia, in October 2016.
Financial support and sponsorship
The study was funded by the Ministry of Research, Technology, and Higher Education of Indonesia.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Murtagh G, Lyons T, O'Connell E, Ballot J, Geraghty L, Fennelly D, et al.
Late cardiac effects of chemotherapy in breast cancer survivors treated with adjuvant doxorubicin: 10-year follow-up. Breast Cancer Res Treat 2016;156:501-6.
Tulubas F, Gurel A, Oran M, Topcu B, Caglar V, Uygur E. The protective effects of ω-3 fatty acids on doxorubicin-induced hepatotoxicity and nephrotoxicity in rats. Toxicol Ind Health 2015;31:638-44.
Ahmed F, Urooj A, Karim AA. Protective effects of Ficus racemosa
stem bark against doxorubicin-induced renal and testicular toxicity. Pharmacogn Mag 2013;9:130-4.
Arung ET, Kusuma IW, Purwatiningsih S, Roh SS, Yang CH, Jeon S, et al.
Antioxidant activity and cytotoxicity of the traditional Indonesian medicine tahongai (Kleinhovia hospita
L.) extract. J Acupunct Meridian Stud 2009;2:306-8.
Gan LS, Ren G, Mo JX, Zhang XY, Yao W, Zhou CX. Cycloartane triterpenoids from Kleinhovia hospita
. J Nat Prod 2009;72:1102-5.
Zhou CX, Zou L, Gan LS, Cao YL. Kleinhospitines A-D, new cycloartane triterpenoid alkaloids from Kleinhovia hospita
. Org Lett 2013;15:2734-7.
Arung E, Kusuma I, Kim YU, Shimizu K, Kondo R. Antioxidative compounds from leaves of tahongai (Kleinhovia hospita
). J Wood Sci 2012;58:77-80.
Djabir YY, Arsyad MA, Budiarto S. Paliasa Leaf (Kleinhovia hospita
Linn) Extract Alleviates Hepatotoxicity Induced by Chronic Use of High Dose Paracetamol. The Second International Seminar Natural Product, Makassar; 2015.
Djabir YY, Arsyad MA, Taebe B. Hepatoregenerative Effects of Paliasa Leave (Kleinhovia hospita
Linn) Extract Against Paracetamol Overdose. Wallacea International Conference, Makassar; 2009.
Raflizar R, Sihombing M. Dekok Daun Paliasa (Kleinhovia hospita
Linn) Sebagai Obat Radang Hati Akut. Center for Research and Development of Disease Control. Litbang, Indonesia: NIHRD; 2012.
Nagy AC, GulAcsi-BArdos P, CserEp Z, Hangody L, Forster T. Late cardiac effect of anthracycline therapy in physically active breast cancer survivors – A prospective study. Neoplasma 2017;64:92-100.
Octavia Y, Tocchetti CG, Gabrielson KL, Janssens S, Crijns HJ, Moens AL. Doxorubicin-induced cardiomyopathy: From molecular mechanisms to therapeutic strategies. J Mol Cell Cardiol 2012;52:1213-25.
Wu R, Gao JP, Wang HL, Gao Y, Wu Q, Cui XH. Effects of fermented Cordyceps sinensis
on oxidative stress in doxorubicin treated rats. Pharmacogn Mag 2015;11:724-31.
Panis C, Herrera AC, Victorino VJ, Campos FC, Freitas LF, De Rossi T, et al.
Oxidative stress and hematological profiles of advanced breast cancer patients subjected to paclitaxel or doxorubicin chemotherapy. Breast Cancer Res Treat 2012;133:89-97.
Rashid S, Ali N, Nafees S, Ahmad ST, Arjumand W, Hasan SK, et al.
Alleviation of doxorubicin-induced nephrotoxicity and hepatotoxicity by chrysin in Wistar rats. Toxicol Mech Methods 2013;23:337-45.
Salouege I, Ben Ali R, Ben Saïd D, Elkadri N, Kourda N, Lakhal M, et al.
Means of evaluation and protection from doxorubicin-induced cardiotoxicity and hepatotoxicity in rats. J Cancer Res Ther 2014;10:274-8.
Koti BC, Nagathan S, Vishwanathswamy A, Gadad PC, Thippeswamy A. Cardioprotective effect of Vedic Guard against doxorubicin-induced cardiotoxicity in rats: A biochemical, electrocardiographic, and histopathological study. Pharmacogn Mag 2013;9:176-81.
Shiromwar SS, Chidrawar VR. Combined effects of p-coumaric acid and naringenin against doxorubicin-induced cardiotoxicity in rats. Pharmacognosy Res 2011;3:214-9.
Ichikawa Y, Ghanefar M, Bayeva M, Wu R, Khechaduri A, Naga Prasad SV, et al.
Cardiotoxicity of doxorubicin is mediated through mitochondrial iron accumulation. J Clin Invest 2014;124:617-30.
Kumral A, Giris M, Soluk-Tekkesin M, Olgaç V, Dogru-Abbasoglu S, Türkoglu Üet al.
Effect of olive leaf extract treatment on doxorubicin-induced cardiac, hepatic and renal toxicity in rats. Pathophysiology 2015;22:117-23.
Elsherbiny NM, Salama MF, Said E, El-Sherbiny M, Al-Gayyar MM. Crocin protects against doxorubicin-induced myocardial toxicity in rats through down-regulation of inflammatory and apoptic pathways. Chem Biol Interact 2016;247:39-48.
Ramesh P, Subramanian S. Flavonoids of Kleinhovia hospita
. Med Educ Res 1984;10:76-7.
Mo JX, Bai Y, Liu B, Zhou CX, Zou L, Gan LS. Two new cycloartane triterpenoids from Kleinhovia hospita
. Helv Chim Acta 2014;97:887-94.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]