|Year : 2012 | Volume
| Issue : 3 | Page : 404-410
Anti-tumor effect of Ardisia crispa hexane fraction on 7, 12-dimethylbenz[α]anthracene-induced mouse skin papillomagenesis
Hamizah Sulaiman, Roslida Abdul Hamid, Yeong Looi Ting, Fezah Othman
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia
|Date of Web Publication||17-Nov-2012|
Roslida Abdul Hamid
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor
Source of Support: None, Conflict of Interest: None
Context: Ardisia crispa Thunb. A. DC (Myrsinaceae) or locally known as hen's eyes has been used in local folk medicine as a remedy in various illnesses. Previously, it has been reported to inhibit various inflammatory diseases. However, research done on this plant is still limited.
Aims: In the present study, the hexane fraction of the A. crispa root (ACRH) was evaluated on the peri-initiation and promotion phases of skin carcinogenesis.
Materials and Methods: This two-stage skin carcinogenesis was induced by a single topical application of 7,12-dimethylbenz(α)anthracene (DMBA) and promoted by repeated treatment with croton oil for 10 weeks in Imprinting Control Region (ICR) mice. Morphological observation would be conducted to measure tumor incidence, tumor burden, and tumor volume. Histological evaluation on the skin tissue would also be done.
Results: The carcinogen control group exhibited 66.67% of tumor incidence. Although, in the ACRH-treated groups, at 30 mg/kg, the mice showed only 10% of tumor incidence with a significant reduction (P < 0.05) in the values of tumor burden and tumor volume of 2.00 and 0.52 mm 3 , respectively. Furthermore, the result was significantly lower than that of the carcinogen and curcumin control. At 100 mg/kg, ACRH showed a comparable result to carcinogen control. On the contrary, at 300 mg/kg, ACRH exhibited 100% tumor incidence and showed a significant elevated (P < 0.05) value of tumor burden (3.80) and tumor volume (14.67 ± 2.48 mm 3 ).
Conclusions: The present study thus demonstrates that the anti-tumor effect of the chemopreventive potential of ACRH is at a lower dosage (30 mg/kg bwt) in both the initiating and promotion period, yet it exhibits a promoting effect at a higher dosage (300 mg/kg bwt).
Keywords: Anti-tumor, Ardisia crispa, croton oil, DMBA, papillomagenesis
|How to cite this article:|
Sulaiman H, Hamid RA, Ting YL, Othman F. Anti-tumor effect of Ardisia crispa hexane fraction on 7, 12-dimethylbenz[α]anthracene-induced mouse skin papillomagenesis. J Can Res Ther 2012;8:404-10
|How to cite this URL:|
Sulaiman H, Hamid RA, Ting YL, Othman F. Anti-tumor effect of Ardisia crispa hexane fraction on 7, 12-dimethylbenz[α]anthracene-induced mouse skin papillomagenesis. J Can Res Ther [serial online] 2012 [cited 2019 Nov 13];8:404-10. Available from: http://www.cancerjournal.net/text.asp?2012/8/3/404/103521
| > Introduction|| |
In today's developed world, the problem of cancer is rapidly increasing and has now become the second cause of death after cardiovascular disease. According to a report released in early 2008, nearly 70,000 new cancer cases were diagnosed among Malaysians, in Peninsular Malaysia, between 2003 and 2005. 
Many scientists have focused on the hazardous effects of various types of cancer treatments, including chemotherapy, radiotherapy, and surgical operations. However, chemotherapy drugs have been found to be far from adequate in providing safety to cancer patients.  Some anticancer drugs have been reported to induce neoplastic tumors in some of the experimental animals.  Radiotherapy, which uses various types of ionizing radiation to destroy the malignant tumor, usually results in severe damage of the normal cells and organs that are exposed to radiation. ,
Today, the chemopreventive approach against cancer development is an important strategy to control the process of cancer induction. This condition leads to the need for exploring traditional medicinal plants or other natural agents that can work as chemopreventive agents. Chemoprevention refers to the administration of chemical agents to prevent the initiational and promotional events that occur during the process of neoplastic development. 
Currently, several plant-derived compounds have been successfully employed in cancer treatment. The most significant example is the compound isolated from the periwinkle Catharanthus Roseus, which belongs to the Vinca alkaloid family. Irinotecan, etoposide, and pactitaxel are the other examples of plant-derived compounds that show impressive anti-tumor activity. 
In recent times, there has been a growing interest in investigating the effects of chemopreventive agents on the inhibition of cancer cell growth. in combination with chemotherapeutics or other common therapies. The number of publications regarding potentiated anti-tumor effects of cancer therapies by chemopreventive agents has dramatically increased in 2005, suggesting that novel combination treatments with common cancer therapies and chemopreventive agents are beginning to receive greater attention in cancer research.
Ardisia crispa has been used in traditional medicine by the Malay community, where its roots and leaves are commonly used. The boiled concoction of the root is reported to be useful for treating earache, cough, fever, diarrhea, throat and chest pain, and also to wash 'dirty blood' or in women dysmenorrheal (menstrual pain). , Previously, Ardisia crispa had been reported to exhibit anti-hypertensive, anti-platelet aggregating factor properties, anti-inflammatory, anti-hyperalgesic, anti-fungal, and anti-pyretic effects. ,,,,, Ardisia crispa has also been reported to possess a chemopreventive effect on the promotion period on DMBA-induced skin papillomagenesis,  but in the present study, we have tested the effect of A. crispa on a modified method of skin carcinogenesis in both the initiation and promotion periods, to confirm its anti-tumor effect when applied in both phases.
Inflammation and wound healing are closely associated with the tumor promotion stage of cancer.  Referring to the beneficial effects of the component inside the plant extract and the plant per se, in inflammation and other ailments, the role of the plant extract was speculated in the amelioration of cutaneous carcinogenesis.  As the n-hexane extract of the Ardisia crispa root has been reported to possess anti-inflammatory properties, as well as a chemopreventive effect,  the present study is conducted to further assess the inhibitory effect of the n-hexane extract of the Ardisia crispa root (ACRH), against the initiation and promotion of carcinogenesis by DMBA and croton oil, respectively, on a two-stage model of skin papillomagenesis in mice. In vivo models of multistage mouse skin papillomagenesis have proven useful in elucidating the molecular events that occur during tumor induction.
| > Material and Methods|| |
Roots of Ardisia crispa were collected from Machang, Kelantan, Malaysia. A voucher specimen was deposited in the herbarium of the Universiti Kebangsaan Malaysia (UKM), Bangi (20841). The roots were washed and dried before they were chopped into pieces and dried in an oven at 40 - 42°C, for three days. Then, the dried roots were ground into a powder form by using a laboratory mill. The powder forms of the A. crispa root were weighed before being macerated in 90% aqueous ethanol. The mixture was then filtered and concentrated, to remove the solvent, by using the rotary evaporator at 40°C, under reduced pressure. The crude ethanol extract was then fractionated by soaking in n-hexane solvent for four days. The mixture was filtered using a filter paper to remove the insoluble particles. Following that, the filtrate was evaporated by using a rotary evaporator at 45°C, under reduced pressure, to remove the n-hexane solvent. The concentrates was dried at room temperature and tested for their anti-carcinogenic properties.
Sixty ICR male mice, aged six to eight weeks were housed in cages (with 10 mice per cage) under normal laboratory conditions of humidity, temperature (25 ± 4°C), and light (12/12-hour light-dark cycle). The mice were allowed one week as an adaptation period, with free access to water and food ad libitum. All procedures were reviewed and approved by the Head of Animal Care and Use Committee (ACUC No: UPM/FPSK/PADS/BR-UUH/00315).
7,12-dimethylbenz(α)anthracene, acetone, and curcumin were purchased from Sigma-Aldrich (USA). Croton oil was purchased from TCI Chemical (Japan). All other reagents were commercially available. DMBA, as a tumor initiator, was dissolved at a concentration of 100 μg/100 μl in acetone. Croton oil, which served as tumor promoter, was dissolved in acetone to give 1% croton oil solution. Curcumin, used as positive control, was dissolved in acetone at a dose of 10 mg/ kg.
High performance liquid chromatography (HPLC) profiling was performed using the HPLC apparatus. The HPLC chromatographic method was successfully developed using a reverse phase column with UV detection at 288 nm, having methanol of HPLC grade as the mobile phase, to resolve 11 peaks from ACRH.
High performance liquid chromatography analysis was done according to the method of Shelar et al. (2009), with slight modification. The HPLC system consisted of an Agilent 1290 series Ultra Performance Liquid Chromatography (UPLC) equipped with an auto sampler, a diode array detector (DAD), and a reverse phase analytical column (HyperClone 3uODS C18, 3 μm, 4.0 × 125 mm, Phenomenex, USA). The mobile phase consisted of Methanol (A), and 0.1% TFA (B) (in proportion of 88: 12 v/v) was degassed before use. The flow-rate was kept at 1.0 ml/minute, temperature of the column was set at 29.99°C ± 1°C, and the injection volume was 20 μl. Detection was carried out at 288 nm.
The Ardisia crispa root was compared with the standard identified compound, AC-2, obtained from the previous report by Roslida.  AC-2, isolated from the Ardisia crispa root, was provided by Dr. Roslida Abdul Hamid. The compound was previously characterized via GC-MS and NMR and identified as 2-methoxy-6-undecyl-1, 4 -benzoquinone [Figure 1].
|Figure 1: Chemical structure of AC-2 identifi ed as 2-methoxy-6- undecyl-1,4-benzoquinone|
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AC-2 (yellow amorphous plates); m.p: 78-80 o C, UV (lmax , MeOH, nm: (265.6, 274.6, 321.0, 361.2; IR (γmax , KBr, cm -1 ): 1510-1560 (C = O conjugated with C = C), 1320, 1360 (C = C); 1H NMR (CDCl 3 , 400 MHz, d): 0.86-0.89 (t, 3H, H-11'), 1.25-1.51 (m, 8H, H-3'- H-10'), 1.62 (s, 1H, H-2'), 2.43 (t, 1H, H-1'), 3.82 (s, 3H, OCH 3 ), 5.87 (d, 1H, H-3), 6.48 (d, 1H, H-5); 13C-NMR (CDCl 3 , 400 MHz, d): 14.1 (s, CH3, C-11'), 22.7 (s, 1C, C- 2'), 27.7-29.7 (m., 8C, C-2'~ C-9'), 31.9 (s, 1C, C-1'), 56.2 (s, 1C, OCH 3 ), 107.1 (s, 1C, C-3), 132. 9 (s, 1C, C-5), 147.6 (s, 1C, C-6), 158.8 (s, 1C, C-2), 182.1 (s, 1C, C = O, C-1), 187.7 (s, 1C, C = O, C-4); GC-MS (m/z, %): 292 (M + , 30), 193 (9), 179 (19), 154 (100), 124 (12), 69 (27), 41 (35).
The in vivo study used the model of two-stage carcinogenesis, according to the method described by Itoigawa et al. In this experiment, the effect of the topical application of ACRH, prior to DMBA application, and further applied before every application of croton oil, for 10 weeks, was evaluated. Sixty ICR male mice were divided into six groups with 10 mice per group. The hair on the dorsal skin of all the animals, in the dorsal area of 1 cm off the tail, was shaved by using an electrical clipper.
Animals in Group I, II, and III received daily topical application of ACRH prior to a single topical application of DMBA treatment (100 μg/100 μl/mouse), and it was further applied every day for a seven-day period. In the promotion phase, ACRH was given 30 minutes before the application of croton oil (100 μ of 1% of croton/mouse), twice a week, for 10 weeks. The doses received were 30, 100, and 300 mg/kg bwt, respectively. Group IV (curcumin) received the same treatment, except that they received a topical application of 10 mg/kg bwt curcumin instead of ACRH. Mice in Group V (carcinogen control) received a similar treatment of DMBA and acetone, without any plant extract. While, Group VI (vehicle control) only received a topical application of acetone throughout the entire experiment.
Weekly observation was carefully performed to count and record the incidence of skin tumor, number of tumor-bearing mice, tumor burden, and tumor volume. The tumor that persisted for at least two consecutive weeks was identified as a skin tumor and included in the cumulative counts. The data was expressed as a percentage of tumor incidence (number of tumor-bearing mice) and tumor burden (number of tumors per tumor-bearing mice).  Tumor volume was calculated using the formula given herewith. 
Tumor Volume (mm 3) = π / 6 × Length × Width × Height
The experiment was terminated at the end of the tenth week of tumor promotion and the mice were sacrificed for histopathological analysis. Skin samples obtained from dissection were fixed in 10% formalin before being processed in an automatic tissue processor, by standard protocols. The processed tissues were embedded in paraffin wax, sectioned with microtome at a thickness of 4 μm and stained with the Hematoxylin and Eosin (H and E) stain, using the routine protocol. The stained slides were observed under light microscope and digital micrographs of the slides were taken.
All data were statistically analyzed by one way analysis of variance (ANOVA), with covariance, followed by the LSD multiple comparison test, to assess the significant differences of mean between the groups. The SPSS 16.0 software was used for the calculation and all values were expressed as mean ± S.E.M (standard error mean). All the values of P < 0.05 were considered as statistically significant.
| > Results|| |
Grounded A. crispa root of 1106.84 g was used for the extraction of aqueous ethanol (70%). Ethanol extraction of A. crispa (ACRE) yielded 170.17 g of extract. Next, ACRE was further fractioned with hexane, which finally yielded 92.09 g of n-hexane extract from the A. crispa root.
The HPLC 'fingerprints' of ACRH showed major peaks at the retention times (minutes) of 3.303, 4.701, and 5.444, respectively, at a wavelength of 288 nm [Figure 2]. The major peak at R t , 3.303, was believed to be benzoquinone (phenolic), , and the two other minor peaks at R t , 4.701 and 5.44, were postulated as triterpenoid saponins, based on the fractionation and isolation done by Roslida,  previously. ACRH chromatograms were compared with AC-2 isolated from the previous report by Roslida.  AC-2 was identified as 2-methoxy-6-undecyl-1,4-benzoquinone [Figure 1]. The difference of the retention time (R t ) shown in both the major peaks [Figure 2] might infer that the compound in the present finding was not similar to AC2, but it might be similar to AC7-1. 
|Figure 2: HPLC chromatogram of (a) ACRH and (b) AC-2 as a standard identifi ed compound from ACRH|
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[Table 1] depicts the findings of the present investigation. The gain in body weight was not affected by the application of ACRH [Table 1]. Tumors started to appear on the treated dorsal skin from five to ten weeks of promotion [Figure 3]. Animals in the carcinogen control group (Group V) showed that 66.67% of the mice developed skin tumors (percentage of tumor incidence). The average number of tumors per tumor-bearing mice (tumor burden) and tumor volume were 2.5 and 9.07 ± 2.13 mm 3 , respectively. The animals in Group IV, curcumin 10 mg/kg (positive) control group, showed 50% tumor incidence. The values of tumor burden and tumor volume were significantly lower (P < 0.05), 2.20 and 1.19 ± 0.73 mm 3 , respectively. Animals in Group I (30 mg/ kg ACRH), exhibited 10% tumor incidence with 2.00 tumor burden and 0.52 mm 3 tumor volume, respectively, which was found to be significantly lower (P < 0.05) than the negative and positive control. On the contrary, in Group II (100 mg/kg ACRH), the tumor incidence and tumor burden were significantly increased to 88.89% and 1.63, respectively [Figure 4] and [Figure 5]. These were significantly higher (P < 0.05) when compared to the positive control (Group IV), but insignificant when compared to the carcinogen control (Group V). At a higher dosage of 300 mg/kg (Group III), ACRH showed its tumor promoting effect by the highest value of tumor incidence (100%), and the elevated values of tumor burden and tumor volume, of 3.80 and 14.67 ± 2.48 mm 3 , respectively. These values were significantly (P < 0.05) different when compared to both the positive and negative control.
|Figure 3: Variation in the percentage of tumor bearing mice during DMBA-induced skin papillomagenesis without or withour A. crispa hexane fraction (ACRH) administration|
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|Figure 4: Variation in the tumor burden during DMBA-induced skin papillomagenesis with or without A. crispa hexane fraction (ACRH) adminstration|
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|Figure 5: Variation in the tumor volume during DMBA-induced skin papillomagenesis with or without A.crispa hexane fraction (ACRH) administration|
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|Table 1: Chemopreventive action of n-hexane extract of Ardisiacrispa root (ACRH) on DMBA – croton oil induced papillomagenesis in mice skin|
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Based on the morphological observations done, the skin tumors, which grew on the dorsal area of the animals in Group III (300 mg/kg bwt), were larger in size and more in number compared to the other groups [Figure 6]. The appearance of the tumors in this group was mostly pedunculated and they had a polypoidal appearance. Animals in Group I (30 mg/kg bwt) showed a significantly lower number of tumors that were smaller in size compared to those in the curcumin (positive) control group. No tumor was observed in the acetone (vehicle) control group.
|Figure 6: The representative from each group at the end of the experiment. (a) Group I, (b) Group II, (c) Group III, (d) Group IV, (e) Group V and (f) Group VI|
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[Figure 7] depicts the histological observation of the effect of treatment with ACRH compared to the positive and negative control on two-stage skin carcinogenesis in mice. Group I (A) and Group IV (D) showed slight epidermal hyperplasia with a few keratin pearls. Group II (B) revealed almost similar observations as Group V. The thickness of hyperplasia was seen to be more in Group III, with a lot of keratin pearl clearly observed (C).
|Figure 7: Representative section of cutaneous tissue from (a) Group I, (b) Group II, (c) Group III, (d) Group IV, (e) Group V and (f) Group VI at the end of the experiment. The one head arrow indicates the keratin pearl. Two head arrow indicates the hyperplasia|
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| > Discussion|| |
In the present study, we have examined the effect of A. crispa root hexane fraction (ACRH) on DMBA-initiated and croton oil-promoted tumorigenesis in a two-stage skin carcinogenesis model. It is known that a single topical application of DMBA (carcinogen) initiates papillomagenesis, which is essentially irreversible, and is followed by treatment with croton oil, which promotes the development of the visible tumor stage.  DMBA treatments generate lipid peroxidation (LPO) and reactive oxygen species (ROS) in the affected areas of the skin, which ultimately lead to the process of carcinogenesis.  There has been no significant difference in weight gain in the experimental group throughout the duration of the experiment (i.e. 10 weeks). Thus, it can be concluded that the ACRH treatment does not influence the normal growth and development of the animals during the experimental period.
The present findings exhibited the same scenario where the carcinogen control group (Group V) exhibited 66.67% tumor incidence after being promoted with croton oil for 10 weeks, after a single application of DMBA. This was perhaps due to deficient natural anti-oxidant defenses, which led to the production of free ROS that has been implicated in the pathogenesis of a wide variety of clinical disorders. , Detrimental effects caused by free radicals occurred as a consequence of an imbalance between the formation and inactivation of these species. , In many previous studies, 100% of tumor incidence was exhibited at the end of the experiment. However, in this current study, only 66.67% of tumor-bearing mice developed at the end of this study. This may be due to the time limitation, which allowed only a short duration of period (10 weeks of promotion period) compared to other long-term studies, which were usually done in at least 20 weeks of time.
On the other hand, the animals in Group 1 (30 mg/kg bwt ACRH) showed a significant reduction (P < 0.05) in the percentage of tumor incidence, delayed onset of tumor latency, and a marked reduction in tumor burden and tumor volume. Surprisingly, the reduction seen was significantly lower than that in the positive (curcumin) control group. This phenomenon showed that a low dose of ACRH (30 mg/kg) exhibited anti-tumor promoting activity. The preventive properties demonstrated by the lowest dose of ACRH tested might indicate that the plant could have acted as an anti-inflammatory agent, as reported by Roslida and Kim,  in inhibiting the DMBA-croton oil-induced mice skin papillomagenesis. Literature suggests that naturally occurring substances have been known to cause inhibition of tumorigenesis, either by preventing the formation of active carcinogen from their precursors or by suppressing the expression of neoplasia. ,
Although the exact mechanism underlying the anti-inflammatory action of ACRH has not been ascertained, an earlier study on its HPLC fingerprinting profiles has shown the major peaks that may contain the benzoquinonoid (phenolic) compound. Furthermore, another benzoquinonoid, AC7-1, also isolated from Ardisia crispa, identified as 2-methoxy-6-tridecyl-1,4-benzoquinone, has been reported to have anti-metastatic and anti-tumor effects in various in-vitro and in-vivo metastasis assays.  Interestingly, both AC-2 and AC7-1 possess a similar skeleton structure (1,4-benzoquinone) with a different number of alkyl groups in the benzene rings. Thus, it may be inferred that the benzoquinonoid compound, whether it is AC-2 or AC7-1., in the ACRH, could have played a synergistic role in the inhibition of papillomagenesis, as observed in the present study.
Similar depletion of papillomagenesis, owing to the inhibition of epidermal ornithine decarboxylase, epidermal DNA synthesis, and promotion of skin tumors by curcumin has been reported earlier, which has led to the usage of curcumin extract as a positive control.  Curcumin is a polyphenol, which can generate reactive oxygen species as a pro-oxidant agent in the presence of copper ions in cells, resulting in DNA damage and apoptotic cell death. , Huang et al. reported that curcumin is able to inhibit the COX activity in the skin and suppress skin cancer development in mice, in the tongue, and colorectal cancer. ,,
In contrast, at a higher dose, Group III, which received topical application of 300 mg/kg bwt ACRH, displayed a promising tumor promoting effect. This has been shown by the early latency period of tumor formation at week five. Animals in this group also showed significantly increased (P < 0.05) tumor incidence, tumor burden, and tumor volume, compared to both the positive and negative control. This might be due to the higher dosage, which could produce toxic effect, thus leading to the promotion of the tumor development on the mice skin.
The general belief is that herbs are safer than synthetic pharmaceutical products, because they are natural. However, the real fact is healing herbs are neither completely safe nor poisonous. They may be effective in low amounts, while in the right amounts they may prove beneficial. Their use in high quantities and over prolonged periods proves to be injurious.  Most phytochemicals with chemopreventive ability have direct antioxidant activity, but many of them can also induce oxidative stress within the cells, when applied at high doses. , In addition ACRH has been shown to possess significant antioxidant activity as well (data not shown).
In recent times, there have been evidences indicating that chemopreventive agents can be used not just to prevent cancer, but also to treat cancer.  Even, the molecular targets of chemopreventive agents are similar to those currently being used for cancer treatment. Therefore, the effect of ACRH at a lower dose, as a chemopreventive, might be beneficial when used in combination with chemotherapeutic agents, to enhance the effect at a lower dose, thus minimizing chemotherapeutic-induced toxicity. As cancer is primarily a disease of old age, less toxic therapy is a major priority.
| > Conclusion|| |
The result of the current study indicated the anti-tumor effect of chemopreventive potential of ACRH at a lower dosage (30 mg/kg). When given continuously in a lower dose (30 mg/kg bwt), ACRH not only lowered the carcinogenicity of DMBA, but also modulated the effect of croton oil as the promoter. It was able to inhibit the initiation of tumor formation, delay the onset of tumor formation, and reduce the percentage of tumor incidence, and there was a marked decrease in tumor burden and tumor volume. However, this extract could also cause a toxicity effect, which might promote tumor development when given in higher dosage (300 mg/kg bwt). The present study demands additional research, to explore the exact mechanism, therapeutic effect, and clinical applicability of A. crispa having an anti-tumor effect of a chemopreventive agent, which may also be used as an adjunct for chemotherapeutic purpose.
| > Acknowledgment|| |
This research was supported by a grant from the Ministry of Higher Education, Malaysia (grant number- 02-04-10-851FR), for scientific development. The authors were equally involved in the research and preparation of this manuscript.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]