|Year : 2014 | Volume
| Issue : 5 | Page : 531-537
Chemopreventive potential of Apium leptophyllum (Pers.) against DMBA induced skin carcinogenesis model by modulatory influence on biochemical and antioxidant
biomarkers in Swiss mice
Himanshu Bhusan Sahoo1, Dev Das Santani2, Rakesh Sagar3
1 NIMS Institute of Pharmacy, NIMS University, Jaipur, Rajasthan, India
2 Department of Pharmacology, NIMS Medical College, NIMS University, Jaipur, Rajasthan, India
3 Department of Pharmacognosy and Phytochemistry, Vedica College of Pharmacy, Bhopal, Madhya Pradesh, India
|Date of Submission||12-Apr-2014|
|Date of Decision||03-Jun-2014|
|Date of Acceptance||20-Aug-2014|
|Date of Web Publication||11-Sep-2014|
Department of Pharmacognosy and Phytochemistry, Vedica College of Pharmacy, Bhopal, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
Aim: The study was designed to investigate the chemopreventive potential of flavonoidal fractions of Apium leptophyllum fruits (FFALF) on Swiss mice.
Materials and Methods: Skin tumor or papilloma was developed by topical application of DMBA (25 μg in 0.1 ml acetone) on intrascapular region of mice, twice weekly for 8 weeks. The animals were divided into six groups: Group I (vehicle control); group II (FFALF control, 5 mg/kg); group III (carcinogenic control, DMBA treated initially for 8 weeks); and group IV, V and VI as pre-treated group (FFALF 5, 10 and 20 mg/kg respectively for 16 weeks along with DMBA treatment). After the 16 th week of treatment; the tumor morphology, skin histopathology, and biochemical and antioxidant biomarkers were measured and compared with carcinogenic control as well as vehicle control.
Results: The co-administration of FFALF with DMBA-treated groups showed significant (P ≤ 0.001) prevention against skin papilloma and normalized the status of lipid peroxidation with antioxidant biomarkers in a dose-dependent manner as compared to carcinogenic control.
Conclusions: Thus, the present study suggests that the FFALF is non-carcinogenic and has chemopreventive potential on DMBA-induced carcinogenesis in mouse, which may be due to the modulation of cutaneous lipid peroxidation or enhancement of total antioxidant capacity.
Keywords: Apium leptophyllum , antioxidant biomarker, chemoprevention, DMBA, skin papilloma
|How to cite this article:|
Sahoo HB, Santani DD, Sagar R. Chemopreventive potential of Apium leptophyllum (Pers.) against DMBA induced skin carcinogenesis model by modulatory influence on biochemical and antioxidant
biomarkers in Swiss mice. Indian J Pharmacol 2014;46:531-7
|How to cite this URL:|
Sahoo HB, Santani DD, Sagar R. Chemopreventive potential of Apium leptophyllum (Pers.) against DMBA induced skin carcinogenesis model by modulatory influence on biochemical and antioxidant
biomarkers in Swiss mice. Indian J Pharmacol [serial online] 2014 [cited 2020 Jan 27];46:531-7. Available from: http://www.ijp-online.com/text.asp?2014/46/5/531/140587
| » Introduction|| |
Epithelial or skin carcinogenesis involves a multistep process proceeded by initiation, promotion and progression of carcinogens. The interaction of carcinogen and accumulation of genetic events within stem cells lead to a progressively dysplastic cellular appearance and deregulated cell growth or differentiation, activating the oncogenes and finally resulting in skin carcinogenesis.  Skin carcinogenesis is the most commonly diagnosed, surpassing lung, breasts, colorectal, prostate, etc. It is initiated as pre-cancerous lesions with environmental toxins playing a very crucial role in the development of skin carcinogenesis.  It is a major and growing public health-related problem among all new carcinogenic cases diagnosed annually in the world with almost one-third cases originating in the skin. In India, skin carcinogenesis accounts for 1-2% of all carcinogenesis as the majority of the population of the country receive high amounts of UV radiation. 
DMBA (7, 12-dimethylbenz(a) anthracene) is a polycyclic aromatic hydrocarbon, acts as a pro-carcinogen and is an ultimate carcinogen after metabolic activation. It is widely used as an initiator as well as a promoter to induce skin carcinogenesis in rodents. Therefore, DMBA is commonly employed to study the chemopreventive potential of natural and synthetic entities.  Liver is the primary site for biotransformation of xenobiotics and for detoxification process. These detoxifying agents perform a crucial role in the metabolic activation and excretion of carcinogenic metabolites. Measurement of the status of these agents in liver helps to test the chemopreventive efficacy of natural and synthetic entities. Studies have documented that when cells are exposed to carcinogens, the detoxification cascade is stimulated. 
Apium leptophyllum (Pers.) Mull. belongs to the Umbelliferae family, popularly known as Ajmuda. It is distributed throughout India, Sri Lanka, Pakistan, South America, Queensland and some tropical areas. In India, it is an annual herb up to 50 cm height, cultivated in Andhra Pradesh, Gujarat, Madhya Pradesh, Jammu-Kashmir and Karnataka.  In the traditional system of Ayurvedic medicine, the fruit is widely used as an antinephritic, antirheumatic and carminative, and is also beneficial in the prevention of tumor, anorexia, vomiting and colic pain. , In Ethiopian traditional medicine, the leaves are used for the treatment of Mitch (a disease associated with inflammation, sweating and loss of appetite).  The volatile oil from the leaves possesses antimicrobial activity against a broad spectrum of pathogens and fungal strains, and also showed an excellent in vitro radical scavenging activity against DPPH.  Phytochemically, it contains volatile oils, coumarin derivatives, terpene hydrocarbons, phenolics and alkaloids, and is a rich source of flavonoids. , Epidemiological and experimental studies indicate that phytochemicals from A. leptophyllum have anti-oxidative and anti-inflammatory properties, which can inhibit tumor initiation, promotion and progression.  However, to the best of our knowledge there is no scientific report available on the chemopreventive potential of A. leptophyllum fruits (FFALF) till now. Therefore, the present study was undertaken to examine the chemopreventive potential of flavonoidal fraction of FFALF against DMBA induced skin carcinogenesis in Swiss mice.
| » Materials and Methods|| |
Chemicals and Reagents
DMBA, reduced glutathione and nicotinamide adenine dinucleotide, and 1,1', 3, 3'- tetramethoxypropane were obtained from Sigma-Aldrich Chemicals Pvt. Ltd., Bangalore, India. Heparin, thiobarbituric acid (TBA), trichloroacetic acid, 2,4-dinitrophenylhydrazine (DNPH), 5,5'-dithiobis (2-nitro benzoic acid) (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), nitro blue tetrazolium (NBT) and Phenazine methosulphate (PMS) were purchased from Hi-Media Laboratories, Mumbai, India. All other chemicals and solvents used were of analytical grade.
Extraction and Isolation of Flavonoidal Fraction from The Plant Material
The fruits of A. leptophyllum were collected from various places in Bhopal district, Madhya Pradesh, India. Further taxonomic identification was conducted in the Department of Botany, Jiwaji University, Madhya Pradesh, India and the voucher specimen (F/HERB/2010/3405) was deposited in the herbarium for future reference. The collected fruits were cleaned, dried and reduced to coarse powder. Then, accurately 100 g of coarse powder was weighed and defatted with petroleum ether for 48 hours in soxhlet apparatus. After complete extraction, the drug was removed from the assembly, air-dried and filled into the soxhlet assembly for successive extraction with 150 ml of methanol for 48 hours. Then, the respective extract was filtered through Whatman No. 1 filter paper, concentrated and dried under vacuum to obtain the methanolic extract. The methanolic extract was filled in plastic bottle and stored at −20 o C until used. The percentage yield of methanolic extract was 16.4% w/w.
The crude methanolic extract (10 g) was then subjected to column chromatography (Silica gel 120 mesh, 500 g) using solvents i.e. diethyl ether, ethyl acetate, chloroform and n-butanol. The collected fractions were subjected to Shinoda test, followed by thin layer chromatography using benzene: Methanol: Ammonia (9:1:0.1) as the solvent system. The spots were visualized by spraying with ammonia, a reagent specific for flavonoids. Further, the flavonoids were confirmed by TLC using the same solvent system and spraying reagent. The fractions showing positive were pooled together and considered as the total flavonoidal fraction. The total flavonoid fraction was concentrated and evaporated. After complete evaporation, a yellow crystalline powder was obtained and stored in air-tight container for further studies.
Random bred male Swiss mice (6-8 weeks old, weighing 20-30 g) were obtained from the animal house of our Institute. They were maintained under standard laboratory conditions of temperature (22 ± 2 o C), humidity (55 ± 5%) and 12:12 hours light and dark cycle, and were fed synthetic pellet with free access to water. The mice were housed 12 per cage and stabilized for one week prior to the commencement of experiment. Before conducting the experiment, it was approved by the institutional animal ethics committee (Regd. No-1693/PO/a/13/CPCSEA).
Oral Acute Toxicity of Flavonoidal Fraction of FFALF
The animals were fasted overnight prior to the experiment. Different graded doses of flavonoidal fraction were administered orally to animal groups and followed for mortality as per the OECD Guideline 425. The LD 50 of FFALF was found at 320 mg/kg body weight. For evaluation of dose-related chemopreventive activity, the graded doses of FFALF viz. 5 mg/kg, 10 mg/kg and 20 mg/kg body weight were chosen.
Experimental Protocol for Dmba-Induced Skin Carcinogenesis Model
Male Swiss mice (72) were selected by randomization and divided into six groups of 12 mice each. The depilatory cream was applied on the intrascapular region of the mice, three days before the commencement of the experiment. Only the mice having no hair growth were selected for the experimental study. 
- Group I: Vehicle control-received acetone (0.1 ml/mouse), topically on intrascapular region twice in a week and distilled water by oral gavage for 16 weeks
- Group II: FFALF control-received FFALF alone (5 mg/kg/body wt), orally once a day for 16 weeks study period (in the morning)
- Groups III: Carcinogenic control-received DMBA (25 μg in 0.1 ml acetone/mouse), topically twice in a week for 8 weeks.
- Groups IV, V and VI: Test groups-received DMBA individually (25 μg in 0.1 ml acetone/mouse) topically twice in a week for 8 weeks with oral administration of graded doses of FFALF (5, 10 and 20 mg/kg/body wt, respectively) by gastric gavage starting one week before the exposure to the carcinogen as DMBA and then continued for 16 weeks with daily administration at morning time.
The tumor parameter and body weight analysis for each group were observed weekly. At the end of the 16 th week of the experimental period, the morphology of the papillomas was analyzed and compared with the carcinogenic control.
Morphology of Papillomas
Papillomas appearing on the shaven area of the skin were examined at weekly intervals in all the groups. Only those papillomas that persisted more than one week or more, with a diameter greater than 1 mm, were taken into consideration for final evaluation. Skin papillomas that regressed after one week observation were not considered for counting. The following tumor parameters were observed: (1). Body weight: The body weight of the mice was measured weekly; (2). Cumulative number of papillomas: The total number of papillomas that appeared till the termination of the experiment; (3). Tumor incidence: The number of mice carrying at least one tumor, expressed as percentage; (4). Tumor yield: The average number of tumors per mouse; (5). Tumor burden: The average number of tumors per tumor bearing mouse; (6). Size of tumor: The diameter of each tumor. (7). Latency period of tumor formation: The time lag between the application of the carcinogenic inducer and the appearance of tumors. 
Biochemical parameters related to carcinogenic process were analyzed after the termination period from each group of experimental animals. After the 16 th week of treatment, the blood sample was collected from each group in a heparinized tube by retro-orbital plexus. The serum was separated by centrifugation at a speed of 1560 × g for 15 min for liver function tests i.e. serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP), total bilirubin (TB) and gamma-glutamyl transpeptidase (GGT) levels. Then, the skin tissues from mice of different groups were collected carefully after dissection, blotted dry, weighed and homogenized using appropriate buffer in a homogenizer with Teflon glass pestle. The skin homogenate was prepared by centrifuging at 1000 rpm for 5 min and the supernatant was used for assay of lipid peroxidation, superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione. After skin removal, the liver was isolated from the body and weighed individually. Then the liver was homogenized and subjected to biochemical estimation of reduced glutathione, glutathione reductase, glutathione-S-transferase and DT-diaphorase. ,,
At the end of the 16 th week, the skin samples obtained by dissection were fixed in 10% formalin before being processed in an automatic tissue processor. Processed tissues were embedded in paraffin wax, sectioned with rotary microtome at a thickness of 2-3 μm and stained with hematoxylin and eosin. Stained slides of each group were evaluated by Nikon Eclipse E-200 Photomicrographic system (400x). 
Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Student-Newman-Keul's test for multiple comparison to assess the significant differences between the groups. SPSS 16.0 software was used for the calculations, and all values were expressed as Mean ± SD. Values with P ≤ 0.001, P ≤ 0.01 and P ≤ 0.05 were considered statistically significant.
| » Results|| |
Effect of F falf0 on Tumor Parameter in Mice
The tumor parameters of both control and experimental animals are shown in [Table 1]. Body weight of carcinogenic control group was significantly (P ≤ 0.001) decreased as compared to the vehicle control whereas the oral administration of FFALF alone to mice showed no significant difference in body weight as compared to vehicle control. But, after oral administration of FFALF groups with carcinogenic treatment, the body weight of each group was significantly increased as compared to carcinogenic control in a dose-dependent manner.
|Table 1: Effect of FFALF on tumor parameters and body weight by DMBA-induced skin carcinogenesis in mice|
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After termination period, the tumor incidence (100%), tumor yield (6.33) and tumor diameter (3.08 mm) were observed in carcinogenic control group, whereas these tumor parameters were significantly prevented in FFALF-treated groups with carcinogenic treatment in a dose-dependent manner. No tumor formation was found in the treatment group of FFALF only (20 mg/kg) till the termination period. The latency period of tumor formation in carcinogenic control was observed in the sixth week, whereas FFALF at the dose of 20 mg/kg did not show appearance of any papilloma at the end of the 16 th week as shown in [Figure 1]. The present study demonstrated that the oral application of FFALF (5, 10 and 20 mg/kg) showed a significant (P ≤ 0.001) reduction as well as a dose-dependent inhibition in tumor incidence, tumor burden, tumor size, cumulative number of papillomas as compared to the carcinogen control. But FFALF, at the dose of 20 mg/kg showed neither any papilloma generation nor body weight reduction in animals. There were no tumor initiations and an improvement in the body weight of animals was observed in the treatment of FFALF only, which indicates that the FFALF was completely non-carcinogenic.
|Figure 1: Effect of FFALF on the gross appearance of skin morphology after 16th week study. (a) Group-III (Carcinogenic control), (b) Group-IV (FFALF-5mg/kg + DMBA), (c) Group-V (FFALF-10mg/kg + DMBA), (d) Group-VI (FFALF-20mg/kg + DMBA)|
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Effect of FFALF on Hematological Parameter From Blood Serum
The serum enzymes from blood sample such as SGOT, SGPT, ALP, total bilirubin and GGT were estimated from the control as well as treated groups of FFALF as shown in [Figure 2]. These serum parameters were higher in the carcinogenic control as compared to the vehicle control, but the treatment of FFALF without carcinogenic treatment showed a close resemblance of serum parameters with the vehicle control. The administration of FFALF along with carcinogenic treatment has produced a significant (P ≤ 0.001) reversible effect by decreasing the level of serum enzymes and bilirubin from the blood sample of animals, implying that it has preventive effect on serum biochemical enzymes.
|Figure 2: Effect of FFALF on hematological parameters from blood serum in mice after 16th week treatment. Datas were expressed as Mean ± SD, (n = 12); Group II, III and IV Compared Vs Group I (###P ≤ 0.001-a, ##P ≤ 0.01-b, #P ≤ 0.05-c) whereas Group IV, V and VI|
compared Vs Group III (*** P ≤ 0.001-d), ns-Not Significant
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Effect of FFALF on Biochemical Parameter From Skin Homogenate in Mice
The strong oxidation potential was reflected by the fall of the SOD, CAT, GSH and GPx levels and rise of lipid peroxidative level in the carcinogenic control group as shown in [Figure 3]. Oral administration of FFALF only to mice had no such effect as compared to vehicle control. The activities of SOD, CAT, GPx and GSH level were significantly (P ≤ 0.001) decreased in groups treated with FFALF and DMBA. All the doses of FFALF with DMBA treatment normalized the antioxidant status and exhibited a dose-dependent relationship. But, the dose of 20 mg/kg of FFALF showed better prevention against oxidative potential among DMBA inducers in skin tissue.
|Figure 3: Effect of FFALF on biochemical parameters from skin homogenate by DMBA induced skin carcinogenesis in mice after 16th week treatment. Datas were expressed as Mean ± SD, (n = 12); Student-Newman-Keuls test compared Vs carcinogenic control; Group II, III and IV Compared Vs Group I (###P ≤ 0.001-a, ##P ≤ 0.01-b, #P ≤ 0.05-c) whereas Group IV, V and VI compared Vs Group III (*** P ≤ 0.001-d)|
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Effect of FFALF on Biochemical Parameter From Liver Homogenate in Mice
The level of detoxifying agent (GSH, glutathione reductase, glutathione-S-transferase and DT-diaphorase) from liver homogenate was performed from all animal groups as shown in [Figure 4]. The level of the detoxifying agent was reduced in DMBA induced group as compared to the vehicle control and FFALF-treated alone group. In carcinogenic treatment with the application of FFALF groups, the status of biochemical parameters from liver homogenate was significantly (P ≤ 0.001 and P ≤ 0.01) increased and normalized. But, the mice group treated with FFALF without treatment of carcinogenic inducer showed no significant difference as compared to the control group.
|Figure 4: Effect of FFALF on biochemical parameter from liver homogenate by DMBA induced skin carcinogenesis in mice. After 16th week treatment, the biochemical parameters i.e. GSH, GR, GST, DT-diaphorase level were accessed. Datas were expressed as Mean ± SD, (n = 12); Student-Newman-Keul's test compared Vs carcinogenic control; Group II, III and IV compared Vs Group I (###P ≤ 0.001-a, ##P ≤ 0.01-b, #P ≤ 0.05-c) where as Group IV, V and VI compared Vs Group III (***P ≤ 0.001-d, *P ≤ 0.01-e, *P ≤ 0.05-f. GSH-Reduced glutathione, GR-Glutathione Reductase, GST-Glutathione-s-transferase|
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Effect of FFALF on Histopathological Study of Skin in Mice
Histopathological analysis of skin showed varying degrees of hyperplasia, keratinized pearls and premalignant lesions with rete ridges in the carcinogenic control group as shown in [Figure 5]. The skin epithelium showed multiple papillomas characterized by the development of finger-like projections protruding over the surface and presence of keratinized tissue. Each papilloma consisted of hyperplasic stratified squamous epithelial cells with a central connective tissue core along with a large number of newly formed blood vessels. The outer lining was covered by keratinized tissue with rete edges. The hyperplasic epidermis, keratinized cell and pre-cancerous lesions were less in co-administration of FFALF with DMBA treatment groups as compared to the carcinogenic control. But, only pre-cancerous lesions were found in 20 mg/kg of FFALF with DMBA treatment group. No carcinogenic symptoms were found in FFALF treatment only, indicating that it is non-carcinogenic. All the doses of FFALF treatment provided preventive effect at cellular level against DMBA treatment.
|Figure 5: Effect of FFALF on histopathology of skin tissue in mice at the end of 16th week. (a) Vehicle control, (b) FFALF-5mg/kg only, (c) Carcinogenic Control, (d) FFALF-5 mg/kg + DMBA, (e) FFALF-10 mg/kg + DMBA and (f) FFALF- 20 mg/kg + DMBA. After termination of 16th week, the skin samples obtained from dissection were fixed in 10% formalin. Stained with haematoxylin and eosin and observed by Nikon Eclipse E-200 Photomicrographic system (400x). Arrows shaped hyperplasia of epidermis (black), keratinized pearls (yellow), premalignant lesions at basement membrane (red) and rete ridges (blue)|
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| » Discussion|| |
The molecules or agents that reverse or stop progression of premalignant cells in which damage has already occurred are referred to as chemopreventive agents. Many scientists have been investigating chemopreventive effects from natural substances against skin carcinogenesis. Therefore, the search of natural product became progressively important, which can either block or reverse the process of carcinogenesis and can be developed as a promising anti-cancer agent. Chemopreventive phytochemicals such as carotenoids, terpenoids, phenolic compounds or flavonoids isolated from vegetables, fruits, spices, teas, herbs and medicinal plants have been reported to suppress carcinogenesis. 
In the present study, the carcinogenic control showed 100% tumor formation, which was histopathologically confirmed by the presence of pre-cancerous lesions, including hyperplasia, dysplasia and keratosis. These types of pre-cancerous lesions were reduced dose dependently in co-administration of FFALF with DMBA-treated groups. Again, the results suggest that oral administration of FFALF at a dose of 20 mg/kg completely prevented the tumor incidence or burden in DMBA-treated mouse, probably by inhibiting abnormal cell proliferation during the DMBA-induced carcinogenic period. Oral administration of FFALF had also the ability to restore the status of the biochemical enzymes in DMBA-treated mice, which suggests that FFALF may have inhibited the metabolic activation of carcinogens or assisted the excretion of carcinogenic metabolites of DMBA.
Increase in lipid peroxidation and decrease in antioxidant status were noticed in the skin tissue of DMBA-treated mice. Insufficient antioxidant potential in carcinogenic control is probably due to overproduction of reactive oxygen species, which plays an important role in tumor initiation by facilitating the metabolic effect of carcinogens. , Results of this study confirm that there exists an imbalance between oxidant and antioxidant status in tumor-bearing mice, which was restored by the administration of FFALF. Thus, it is suggested that A. leptophyllum could possibly exert its chemoprevention through antioxidant mechanism. A chemopreventive agent acts by preventing metabolic activation from carcinogen, increasing the detoxification of the carcinogens and blocking the interaction of carcinogen with cellular macromolecules.  The present study suggests that FFALF inhibited the metabolic activation of DMBA and enhanced the excretion of carcinogenic metabolites by stimulating the activities of detoxification cascade in the skin. There exists an inverse correlation between the status of lipid peroxidation and rate of cell proliferation. As the tumor progressed, lipid peroxidation by-products were lowered in tumor cells. Lower lipid peroxidation accompanied by decrease in glutathione and glutathione peroxidase activity were observed in the skin tumor cells, which facilitate cell proliferation and promote growth advantage in tumor cells over the surrounding normal cells. GPx, SOD and CAT form a part of the crucial processes involved in cellular antioxidant defense mechanism whereby peroxides and superoxides are inactivated.  Glutathione peroxidase and its co-substrate glutathione are essential for the maintenance of cellular integrity due to their regulatory effects on cell proliferation. An increase in lipid peroxidation and decrease in antioxidants status (reduced glutathione, glutathione peroxidase, superoxide dismutase and catalase) has been well documented in the plasma of skin cancer patients and rodents bearing skin tumors. , The present study demonstrated that both co-administration and FFALF alone can activate these enzymes during exposure of DMBA.
In conclusion, the present study indicates that FFALF affects the enzyme activities of liver as well as lipid peroxidation and exhibits chemopreventive activity. Particularly at the dose of 20 mg/kg, FFALF was effective in suppressing skin tumor growth, which may be due to anti-lipid peroxidative or antioxidant potential during DMBA tumor induction.
| » Acknowledgement|| |
My sincere thanks to Principal of Vedica College of Pharmacy, Bhopal, India and Dean of NIMS Institute of Pharmacy, NIMS University, Jaipur, India, for guiding and permitting the lab facility for my research work.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]