Therapeutic Potential of Standardized Extract of Melilotus indicus (L.) All. and Its Phytochemicals against Skin Cancer in Animal Model: In Vitro, In Vivo, and In Silico Studies.

Melilotus indicus (L.) All. is known to have anti-inflammatory and anticancer properties. The present study explored the in vivo skin carcinogenesis attenuating potential of ethanolic extract of M. indicus (L.) All. (Miet) in a 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin cancer model. The ethanolic extract of the plant was prepared by a maceration method. HPLC analysis indicated the presence of quercetin in abundance and also various other phytoconstituents. DPPH radical scavenging assay results showed moderate antioxidant potential (IC50 = 93.55 ± 5.59 μg/mL). A topical acute skin irritation study showed the nonirritant nature of Miet. Data for the skin carcinogenic model showed marked improvement in skin architecture in Miet and its primary phytochemicals (quercetin and coumarin) treated groups. Miet 50% showed comparable effects with 5-fluorouracil. Significant (p < 0.05) anticancerous effects were seen in coumarin-quercetin combination-treated animals than in single agent (coumarin and quercetin alone)-treated animals. Chorioallantoic membrane (CAM) assay results showed the antiangiogenic potential of Miet. Treatment with Miet significantly down-regulated the serum levels of CEA (carcinoembryonic antigen) and TNF-α (Tumor necrosis factor-α). Data for the docking study indicated the binding potential of quercetin and coumarin with TNF-α, EGFR, VEGF, and BCL2 proteins. Thus, it is concluded that Miet has skin cancer attenuating potential that is proposed to be due to the synergistic actions of its bioactive molecules. Further studies to explore the effects of Miet and its bioactive molecules as an adjuvant therapy with low dose anticancer drugs are warranted, which may lead to a new area of research.

Introduction

Skin cancer is the most common type of cancer, and its incidence is increasing rapidly all over the world. The literature has shown that out of 18.1 million new cases of cancer, among them 1.04 million belong to skin cancer.[1] However, among all skin cancers, the prevalence of nonmelanoma skin cancer (NMSC) is 20% higher than that of malignant melanoma.[2] In 2016, a national survey estimated that the prevalence of skin cancer seems to be 52.3% in women and 47.7% in men.[3,4] Generally, in men the incidence of cutaneous cancer is about double than of women due to their direct exposure to ultraviolet radiation, polycyclic aromatic hydrocarbons, heavy metals, volatile organic compounds, and polluted environments.[5] Polycyclic aromatic hydrocarbons (PAHs) are dangerous environmental pollutants that play a vital role in the etiology of cancer because these compounds generate peroxide and superoxide anion radicals, which induce oxidative stress in form of lipid peroxidation.[6] DMBA (7,12-dimethylbenz[a]anthracene) is a PAH that initiates skin cancer in a short time because it can cause DNA damage.[7] DMBA is metabolized by two enzymes CYP1A1 and CYP1B1 and then converted to epoxide-3,4-diol DMBA. This metabolite form DMBA–DNA adduct activates proto-oncogene or inactivates tumor suppressor genes that will initiate tumor formation.[8] Due to the growing incidence of skin cancer and the higher ratio of side effects caused by marketed anticancer drugs, it is direly needed to develop natural anticancer drugs possessing fewer side effects. Plants have been consumed as “medicinal agents” since the origin of mankind because of their general acceptance, economical benefits, and multistep targeting potential toward cellular signal transduction pathways.[9]

Melilotus indicus (L.) All. is a small herb of the family Fabaceae. It has been traditionally used as an antispasmodic, emollient, analgesic, and astringent agent since ancient times.[10] Data of several in vitro studies revealed that M. indicus (L.) All. shows cytotoxic activity against different cancerous cell lines due to its various properties.[11] The findings of our previous studies showed that ethanolic extract of M. indicus (L.) All. and various other traditional plants and their active constituents have potent anti-inflammatory and ulcer healing properties.[12−16] However, to the best of our knowledge, there is no study available that reports the anticancer effects of M. indicus (L.) All. against DMBA-induced skin cancer. Therefore, the present study was designed to evaluate the antiskin cancer efficacy of M. indicus (L.) All. extract.

Results

DPPH (1,1-Diphenyl-2-picrylhydrazyl) Scavenging Assay

The results of the DPPH radical-scavenging assay showed that M. indicus (L.) All. ethanolic extract possessed moderate to weak radical scavenging capacity with an IC50 value of 93.55 ± 5.59 μg/mL. The IC50 value of ascorbic acid was calculated to be 10.08 ± 3.99 μg/mL.

Quantitative Analysis of M. indicus (L.) All. Ethanol Extract by HPLC

Total nine compounds were identified in Miet, i.e., quercetin (7.63 ppm), gallic acid (0.43 ppm), caffeic acid (1.44 ppm), vanillic acid (5.62 ppm), benzoic acid (5.42 ppm), chlorogenic acid (5.92 ppm), syringic acid (1.59 ppm), p-coumaric acid (0.56 ppm), and m-coumaric acid (0.36 ppm) (Figure , Table ). Structures of a few important phytoconstituents have been drawn and are provided in Figure .

Figure 1

HPLC chromatogram of M. indicus (L.) All. Ethanolic extract compounds were identified by comparing the retention time with standards.

Table 1

List of Compounds Analyzed by HPLC

no.	compd name	tR (min)	% area	quantity (ppm)
1	quercetin	2.873	2.3	7.63
2	gallic acid	4.540	0.2	0.43
3	caffeic acid	12.680	0.5	1.44
4	vanillic acid	13.167	1.5	5.62
5	benzoic acid	14.993	0.8	5.42
6	chlorogenic acid	15.800	1.2	5.92
7	syringic acid	16.820	1.0	1.59
8	p-coumaric acid	17.847	0.8	0.56
9	m-coumaric acid	20.053	0.5	0.36

Figure 2

Structures of phytoconstituents present in the ethanolic extract of M. indicus (L.).

Skin Irritation Study

Findings of the skin irritation study showed that topical application of Miet incurred no sign of irritation or toxicity in the rats (Figure S1). No signs of redness, itching, or wounds were observed in the animals treated with Miet. Moreover, no significant changes in the weight of animals were observed at the end of the study (Table S1). Histopathological examination of excised organs showed no cellular abnormalities in Miet-treated animals as compared with healthy control group (Figure S2).

No significant difference was observed in body weight before and after completion of study as shown in Table S1.

Antitumor Study

Effect of Ethanolic Extract of M. indicus (L.) and Active phytochemicals on Changes in Bodyweight (g)

Mean body weights of animals treated with vehicle (group I), Miet 25% (group III), Miet 50% (group IV), and 5-FU (group V) were increased by 6%, 3%, 3%, and 5%, respectively, when compared with initial body weights of respective groups. Body weights of animals in carcinogen-treated (group II) animals were reduced by 15% as compared with initial body weights (Table ).

Table 2

Weight Variations in Different Treatment Groups DMBA-Induced Skin Carcinogenesisa

	body weight (g)
treatment groups	initial	final	percent increase or decrease
Group I (vehicle treated)	32.8 ± 1.92	34.8 ± 1.92	+6.09
Group II (carcinogen treated)	27.8 ± 1.09	23.6 ± 1.14	–15.10
Group III (Miet 25%)	26.2 ± 1.30	27.2 ± 2.59	+3.8
Group IV (Miet 50%)	28.6 ± 2.07	29.6 ± 1.14	+3.49
Group V (standard, 5-FU)	28.6 ± 1.87	30.2 ± 1.48	+5.59
Active Constituents Study
Group I (carcinogen treated)	29.33 ± 1.15	25.66 ± 0.57	–12.51
Group II (coumarin)	30 ± 1.0	26.66 ± 1.15	–11.13
Group III (quercetin)	30.33 ± 1.5	30 ± 2.0	–1.08
Group IV (Q.C)	31 ± 2.64	29 ± 1.0	–6.45

Values shown are Mean ± SD of weight variations in DMBA-induced skin cancer model (n = 6). + sign showing increase in body weight, - sign showing decrease in body weight and Q.C showing quercetin-coumarin combination treated group.

In the compound study, the body weight of Group I (carcinogen treated) after topical application of carcinogen showed significant reduction. The mean body weight of animals in group II (coumarin treated) show 4% reduction, while in group III (quercetin treated) there was no significant effect observed. Animals in group IV (combination treated) show a 2% decrease in body weight as shown in (Table , lower part).

Effect of Ethanolic Extract of M. indicus (L.) on Tumor Yield, Tumor Burden, Cumulative Number of Tumors, and Tumor Incidence

The gradual reduction in tumor yield observed in mice treated with Miet 25% and Miet 50% creams was 3.86 ± 1.86 (p > 0.05), 2.94 ± 1.75 (p < 0.05), respectively, when compared with tumor yield (6.03 ± 3.01) in a carcinogen-treated (DMBA-croton oil) group. Animals treated with 5-fluorouracil (group V, 5 mg/mL) showed the lowest tumor yield of 2.32 ± 1.70 (p < 0.05).

The tumor burden in animals treated with Miet (25% and 50%) and 5-FU (5 mg/mL) was 5.61 ± 2.37, 5.01 ± 2.74 , and 4.33 ± 2.67 (p < 0.05). The tumor burden in carcinogen the control group was observed to be 7.59 ± 2.30. The cumulative number of tumors in carcinogen-treated, Miet 25% and 50%, and 5-FU treated groups was 30.2 ± 15.1, 23.1 ± 11.8, 10.8 ± 5.64, and 8.73 ± 5.78, respectively. A significant decrease (p < 0.05) in the number of tumors was observed in animal groups treated with Miet 50% and 5 mg/mL 5-FU, respectively (Table ).

Table 3

Effect of Ethanolic Extract of M. indicus (L.) All. on Morphological Parameters in DMBA-Induced Skin Carcinogenesis Model

parameters	Group II (carcinogen treated)	Group III (Miet 25%)	Group IV (Miet 50%)	Group V (5-FU)
tumor yield	6.03 ± 3.01	3.86 ± 1.86	2.95 ± 1.75	2.33 ± 1.70
tumor burden	7.59 ± 2.30	5.61 ± 2.37	5.01 ± 2.74	4.33 ± 2.67
tumor incidence	65.5 ± 32.4	50.9 ± 25.9	41.8 ± 24.4	30.9 ± 20.7
cumulative no. of tumors	30.2 ± 15.1	23.1 ± 11.8	10.8 ± 5.64	8.73 ± 5.78

Effect of Phytochemicals (Active Constituents) Alone and in Combination on Tumor Yield, Tumor Burden, Cumulative Number of Tumors, and Tumor Incidence

Similarly, a significantly low (p < 0.05) tumor yield was observed in animals treated with a quercetin–coumarin combination (0.89 ± 0.73) as compared to the carcinogen-treated group (5.81 ± 4.06). The tumor burden in carcinogen control, quercetin alone, coumarin alone, and quercetin–coumarin combination treated groups was 8.12 ± 3.36, 4.73 ± 1.72, 3.42 ± 1.73, and 2.73 ± 1.62, respectively. Moreover, the highest tumor incidence (63.3 ± 24.6) was observed in the carcinogen-treated group, while the quercetin–coumarin combination treated group had the lowest tumor incidence. The cumulative number of tumors in carcinogen control, coumarin alone, quercetin alone, and the quercetin–coumarin combination treatment groups were 34.9 ± 24.4, 16.6 ± 12.0, 8.90 ± 6.03, and 5.40 ± 4.40, respectively. The highest cumulative number of tumors was observed in the carcinogen-treated group, while quercetin alone and the quercetin–coumarin combination treatment groups showed better effects as compared with the rest of the groups (Table ).

Table 4

Effect of Ethanolic Extract of M. indicus (L.) All. on Morphological Parameters in DMBA-Induced Skin Carcinogenesis Model

parameters	Group I (carcinogen control)	Group II (coumarin treated)	Group III (quercetin treated)	Group IV (quercetin–coumarin treated)
tumor yield	5.81 ± 4.06	2.36 ± 1.47	1.48 ± 1.00	0.89 ± 0.73
tumor burden	8.12 ± 3.36	4.73 ± 1.72	3.42 ± 1.73	2.73 ± 1.62
tumor incidence	63.3 ± 24.6	43.3 ± 19.6	36.7 ± 18.9	25.0 ± 18.0
cumulative no. of tumors	34.9 ± 24.4	16.6 ± 12.0	8.90 ± 6.03	5.40 ± 4.40

Carcinoembryonic Antigen (CEA) Analysis

The average serum levels of CEA (ng/mL) in healthy, carcinogen control, Miet (25 and 50%) and 5-FU treated groups were 1.03 ± 0.06, 3.10 ± 0.14, 2.23 ± 0.38, 1.31 ± 0.17, and 1.21 ± 0.17 ng/mL, respectively. CEA levels were significantly high (p < 0.05) in the carcinogen control group as compared to the Miet 25%, Miet 50%, and 5-FU treatment groups (Figure a).

Figure 3

(A) Graphical presentation of CEA (ng/mL) analysis. (a) Carcinogenic compound, MIET 25%, MIET 50%, and % FU, while in (b) CEA (ng/mL) analysis in antitumor study of carcinogenic compound, coumarin, quercetin, Q.C (coumarin, quercetin combination group). Data is expressed by mean ± SD p < 0.01, p < 0.05, p > 0.05.

Average serum levels of CEA (ng/mL) in carcinogen control, coumarin alone, quercetin alone, and the quercetin–coumarin combination treatment group were 3.20 ± 0.42, 2.01 ± 0.02, 1.31 ± 0.16, and 0.99 ± 0.01 ng/mL, respectively. Quercetin-coumarin combination treatment showed significant (p < 0.05) low serum CEA levels as compared with the carcinogen control group (Figure b).

Histopathological Examination

Microscopic Study of Skin Layers

Histopathological examination of skin sections stained with H&E stains showed that the epidermis in normal healthy animals was normal in architecture and was two layered and unremarkable. The underlying dermis showed skin appendages and hair follicles. Skin sections of the carcinogenic group (DMBA + croton oil) showed large keratin pearls, a prominent feature of well-differentiated squamous cell carcinoma. The Miet 25% treated group exhibited thin epidermis and damaged dermis layers and contained large abnormal growth of cells with altered morphology. The Miet 50% treated group showed a dysplastic epithelium with irregular downward proliferation of epithelial cells into the dermis. The 5-fluorouracil treated group showed a two-layer normal epidermis and was unremarkable. The underlying dermis showed nonuniform structure with less differentiated squamous cell carcinoma (Figure ).

Figure 4

Histopathological examination of H&E stained skin section under a microscope at 10× (left side) and 40× (right side). Effect of ethanolic extract of M. indicus (L.): In healthy (Normal Control) group; epidermis is two layered and unremarkable. The underlying dermis shows skin appendages and hair follicles. Ethanolic extract of M. indicus (L.) Miet) 25% treated group shows thin epidermis and damaged dermis layers and contains a large abnormal growth of cells with altered morphology. Miet 50% treated group shows dysplastic epithelium with irregular downward proliferation of epithelial cells into the dermis. 5-Fluorouracil treated group shows two-layered normal epidermis and unremarkable. The underlying dermis shows a nonuniform structure with less differentiated squamous cell carcinoma (SCC). Coumarin treated group shows dysplastic cells of poorly differentiated squamous cell carcinoma with less keratin and bizarrely shaped cells. Quercetin-treated group shows two-layered epidermis and dermis with skin appendages and hair follicles like normal skin histology. The quercetin and coumarin (Q.C) combination group had a wound area, focal ulceration is noted. The underlying dermis shows moderate mixed inflammation predominated by polymorphs. No skin appendages and hair follicles are present. No evidence of malignancy found. Carcinogenic (DMBA + croton oil) group skin section during compound study shows large keratin pearls of well differentiated squamous cell carcinoma (arrow).

Meanwhile, when the effect of quercetin, coumarin, and a combination of both (Q.C) was examined it showed dysplastic, bizarrely shaped cells of poorly differentiated squamous cell carcinoma having less keratin formation in coumarin-treated animals. The quercetin-treated group exhibited two layered and unremarkable epidermis. The underlying dermis showed skin appendages and hair follicles like normal skin histology and cells with less altered morphology. The quercetin–coumarin combination treatment (Q.C) group showed focal ulceration in the wound area, and a scab was present showing necro-inflammatory debris and bacterial colonies. The underlying dermis showed moderate mixed inflammation predominated by polymorphs. No skin appendages and hair follicles were present. No evidence of malignancy was found (Figure ).

Skin Gross Appearance

Skin gross appearance is observed from week 1 to week 17 during the whole disease induction and treatment time period. DMBA treated group showed skin wound, ulceration, lesion production, and skin tumor development from week 6. All of the treatment groups such as 5-Fu, Miet 25, and Miet 50% and active compounds containing both coumarin and quercetin started healing process from week 10, but visible effects were present from week 14 to week 16. 5-FU showed more healing capacity than Miet 50% and 25%, respectively (Figure A), while the coumarin–quercetin combination group effects were better than both compounds alone (Figure a,b).

Figure 5

Gross appearance of skin tumors at the dorsal area of mice. (a) Skin gross appearance from week 6 to 16. Five groups: healthy (normal control), carcinogen (DMBA treated) group, ethanolic extract of M. indicus (L.) Miet 25% and Miet 50% treated groups. Tumor growth started from weeks 6-8, while the peak effect and healing started from week 14 and were visible at week 16. The most prominent healing phenomenon was visible in the 5-FU (5-fluorouracil) group, while MIET 50% effects were better than Miet 25%. (b) Gross appearance of skin tumors at the dorsal area of mice from start of tumor (weeks 6–17) in different groups during compound study of the antitumor activity of DMBA-induced skin carcinogenesis model. Development of tumor in different study groups from quercetin to carcinogen control. After initiation tumor development from week 6 to 8 of study and continued growth in the carcinogenic group until termination of the study, healing started from weeks 12 to 14 and was visible from weeks 16 to 17. Most prominent effects were in the combined (Q.C) coumarin and quercetin group. Normal control was common in both (a) and (b).

Antiangiogenic Effects of Ethanolic Extract of M. indicus (L.) All

The results of the CAM assay showed significant reduction in blood vasculature in Miet 100 and 200 μg/mL treated eggs as compared with the control group (Figure ).

Figure 6

Antiangiogenic effect of ethanolic extract of M. indicus (L.) All. Figure shows comparison of blood vessels growth rate in control and Miet treatment groups at dose of 200 and 100 μg/mL of in chicken chorioallantoic membrane assay. (a) Normal vasculature in control group, (b) showed effect of Miet 100 μg/mL on vascular over growth, (c) showed effect of Miet 200 μg/mL on vascular over growth. Marked reduction in blood vessels over growth was obderved in both the Miet treatedCAMs.

TNF-α ELISA

Serum levels of TNF-α in vehicle treated, carcinogen control, Miet (25% and 50%) and 5-FU treated groups were 303.739, 5729.969, 2595.115, 1175.333, and 436.683 pg/mL, respectively. Significantly high (p < 0.05) levels of TNF-a were observed in the carcinogen control group as compared with the Miet and 5-FU treatment groups (Table ).

Table 5

Effect of Miet Treatment on Serum Levels of TNF-αa

groups	treatment	TNF-α (pg/mL)
Group I (vehicle treated)	acetone topically + double distilled water orally	303.739
Group II (carcinogen treated)	DMBA + croton oil	5729.969
Group III (Miet 25%)	(DMBA + croton oil) + MI extract 25%	2595.115
Group IV (Miet 50%)	(DMBA + croton oil) + MI extract 50%	1175.333
Group V (5-FU)	(DMBA + croton oil) + 5- fluorouracil	436.683

Values shown are mean ± SD of serum levels of TNF-α study (n = 3) where * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and ns = p > 0.05, respectively.

Molecular Docking Study

In the present study, the best-fitted compatibility of two structurally distinct ligands with various molecular targets has been studied using a molecular docking approach. The docking scores (Cscore) of curcumin CUR and coumarin CUM for CD31 protein were 5.89 and 5.16, respectively, which indicates that the compound CUR exhibits slightly higher binding affinity toward CD31 than CUM. Similarly, only a minor difference was observed in the TNF-α-CUR (CScore: 4.58) and -CUM (CScore: 4.15) systems. Since the difference in docking scores of CUR- and CUM-bonded systems is negligible, it clearly reflects that despite its smaller size (Figure ) our test compound CUM exhibits almost similar binding affinity toward CD31 and TNF-α as that observed in the CUR-bonded system.

Figure 9

Chemical structures of standard curcumin (CUR) and tested compound coumarin (CUM). The reactive sites are highlighted with purple and smudge rectangles, where hydrogen bond acceptor (HBA) groups are highlighted as smudge rectangle and hydrogen bond donors (HBD) are highlighted as purple rectangles.

The docking scores of the standard compound CUR for VEGFR, BCL2, and ROS1 are 7.26, 6.78, and 8.32, respectively, which indicates that CUR has relatively higher binding potential toward VEGFR, BCL2, and ROS1 among all CUR–protein complexes (Table S2). Conversely, the compound CUR fail to display significant binding affinity toward VEGFR, BCL2, and ROS1 proteins. However, a nonsignificant difference between the binding affinities of CUR- and CUM-EGFR complexes is observed, which reveals that the compound CUM exhibits moderate binding potency toward EGFR as compared to EGFR-CUR complex. Furthermore, detailed comparison of docking scores and total energies of the top-ranking ligand conformations along with binding site residues involved in H-bond interactions in various ligand–protein systems are tabulated in Table S2.

Discussion

The current study was designed to evaluate the therapeutic potential of topical application of M. indicus (L.) All. ethanolic extract (Miet) and its major phytochemicals (coumarin and quercetin) in the DMBA-induced skin carcinogenesis mice model. Before commencement of the anticancer study, a skin irritation/toxicity study was conducted to evaluate the safety of Miet toward topical use. Cutaneous application of an ethanolic extract of M. indicus (L.) All. showed no visible sign of skin irritation (redness, erythema, etc.), necrosis, or mortality (Figures S1 and S2). In addition, no significant difference in body weights of animals and hematological profile were observed. The findings of this safety study are in line with our previously published work showing a safe therapeutic profile of Miet[14] indicating that M. indicus (L.) All. can be used as an oral and topical therapeutic candidate against multiple pathologies. Epidemiological data revealed that direct exposure to ultraviolet radiations is the main carcinogen involved in the development of both melanoma and nonmelanoma skin cancers globally.[17] In the present research work, dimethylbenz[a]anthracene (DMBA) and croton oil induced skin cancer exhibited the same kind of skin cancer patterns, i.e., a solid thickened skin mass in the form of lesions, at week 6 of the study. However, complete tumor development was seen in week 8 of the study (Figure ) after application of DMBA. In fact, the enzymatic breakdown in the body converts DMBA into 1,2-epoxide-3,4-diol DMBA, which forms an adduct with DNA and is responsible for intracellular genetic mutations; thus, it is considered to be a prerequisite for development of tumor.[8] In addition, reactive oxygen species (ROS) play a prominent role in the initiation and promotion of chemical-induced carcinogenesis. Moreover, DMBA and croton are considered as key factors in the development of abnormally uncontrolled inflammation,[18] which activates COX-2 and prostaglandin production and leads to a cascade of events for papilloma formation.[8] Thus, inhibition of oxidative stress and abnormal DMBA metabolic product synthesis is a promising step for inhibition of tumorigenesis. In the present study, Miet showed moderate radical-scavenging potential, which is suggested to be due to its multiple phytochemicals, i.e., quercetin as well as coumarin.[19] In the present study, tumor-bearing animals showed dose-dependent effects after treatment with Miet 50% and 25%. In addition, levels of tumor yield, tumor burden, cumulative number of tumor, and tumor incidence were significantly decreased in the Miet 50% treatment group and approached almost normal values, hence indicating the excellent therapeutic capability of Miet against skin cancer (Tables and 3). The 5-FU group showed comparable (p < 0.001) anticancerous effects as compared to the healthy group. In histopathological examination, carcinogen control animals exhibited signs of large keratin pearls that is one of the most prominent feature of well-differentiated squamous cell carcinoma (SCC), whereas the 5-FU and Miet treatment groups showed a two-layered normal epidermis and nonuniform dermis structure with less differentiated SCC (Figure ).

Cancer chemoprevention by natural phytoconstituents has emerged as one of the hottest areas of investigation in the recent times.[20] In the current research, an HPLC study of Miet revealed presence of quercetin (a well-known flavonoid) and p-coumaric acid (hydroxycinnamic acid derivative) and various other phytochemicals (Figures and 2). Studies have been shown antioxidant, anti-inflammatory, cytotoxic, and antiangiogenic attributes of quercetin and coumarin along with their noteworthy effects to reduce viability and induce apoptosis in melanoma cells.[21] The chemoprotective effects of quercetin have been documented by a series of investigations. Chinembiri and colleagues analyzed the inhibitory action of quercetin on ROS generation and activated restoration of antioxidant enzymes levels in C141 mouse epidermal cells. Moreover, quercetin inhibits signal transducer and activator components of an oncogene protein transcription-3 (STAT-3), thus showing antimelanoma activities.[22] Coumarin has anti-inflammatory activity due to its high hydroxyl group scavenging capacity and redox action.[23] Therefore, in the present study, quercetin and coumarin, as reported in our previous study,[14] were found to be highest in concentration in M. indicus (L.) All. Ethanolic extract was also explored for its anticancer activity against DMBA-induced cancer. Findings have shown that animals treated with individual compounds (coumarin and quercetin) showed a lower therapeutic effect than the quercetin–coumarin combination treatment group. Other major compounds reported in HPLC analysis were gallic acid, caffeic acid, vanillic acid, benzoic acid, chlorogenic acid, syringic acid, and m-coumaric acid. Many of these have been already approved as free radical scavengers as well as tumor suppresser agents in preclinical studies, while few are in clinical trials.[24] Additionally, a decrease in the expression of TNF-α (pro-inflammatory cytokine) also revealed a profound effect of M. indicus (L.) All. toward chronic inflammation in a dose-dependent manner (Table ).

Phytochemical-enriched plant extracts can slow differentiation and multiplication of cancerous cells and induce apoptosis simultaneously, which halts tumor eradication by hampering angiogenesis and consequently metastasis.[25] Therefore, the antiangiogenic property of Miet was also evaluated through chorioallantoic membrane assay in the current research work. The ethanolic extract of M. indicus (L.) showed a dose-dependent response toward the dispersion of a blood vascular network and thus can be used as an adjuvant therapy in cancer (Figure ). Multiple studies have reported antiangiogenic potentials of quercetin and coumarin by inhibiting proliferation, migration, and tube formation of endothelial cells.[26] Synergistic actions of coumarin, quercetin, and other biologically active Phyto molecules are suggested to contribute to the anticarcinogenic effects of Miet against DMBA-induced skin cancer. A discussion of molecular docking studies which explain the docking of curcumin with BCL2, EGFR, and TNF-α, etc. (Figure S3A–D and Figure S4A–H) and a detailed discussion regarding scoring and in silico bonding is also present in the Supporting Information. Overall, data of ELISA and docking studies propose that treatment with Miet down-regulated the expression of multiple tumor promoting proteins leading to improvement in the macroscopic and microscopic parameters in in vivo skin cancer model.

Conclusion

Altogether, findings of the present study conclude that the multicomponent ethanolic extract of M. indicus (L.) All. possesses antioxidant and antiangiogenic properties which are proposed to be the main contributing factors toward its antiskin cancer attributes. Down-regulation of multiple interlinked tumor promoting cytokines is suggested to be the major molecular event for the observed pharmacological effects. Further studies to evaluate the role of ethanolic extract of M. indicus (L.) All. and its bioactive molecules in the potentiation of anticancer actions and diminution of side effects of standard chemo-drugs as an adjunct therapy are warranted. Moreover, development of stable oral formulations containing standardized M. indicus (L.) extract alone and in combination with low dose chemo-drugs is suggested to be prepared and evaluated for its antiskin cancer actions.

Experimental Section

Collection of Plant Material

M. indicus (L.) All. (Miet) whole plant was collected from local fields in the district of Okara, Punjab, Pakistan and identified by a botanist, Dr. Mansoor, Department of Botany, University of Agriculture Faisalabad, Punjab, Pakistan. The sample was submitted to the herbarium for future reference.[14]

Preparation of Extract

For extraction, a coarse powder of Miet whole plant was macerated in absolute ethanol (99.5%) in an airtight container and was occasionally stirred with a glass rod. The filtrate was evaporated at 40 °C using a rotary evaporator, and the resultant green extract (Miet) was stored in a tightly closed glass bottle at 4 °C.

Determination of Antioxidant Activity

The antioxidant capacity of Miet was evaluated in an in vitro DPPH assay following the reported protocols.[14,27]

High-Performance Liquid Chromatography (HPLC) Analysis

The reported gradient HPLC method was used to screen the phenolic and flavonoid contents of Miet. Two types of mobile phase were used, i.e., mobile phase A, a mixture of water and acetic acid (H2O: AA-94:6) with pH = 2.27, and mobile phase B, acetonitrile (100%). B was at 100% from 0 to 15 min, at 15% from 15 to 30 min, and at 45% and then 100% for 30–45 min. The flow rate was set as 1 mL/min, and absorbance of sample was measured at room temperature using an ultraviolet visible detector set at 280 nm wavelengths. Compounds were identified by comparing the peaks of the reference standard and sample.[28]

Pharmacological Studies

All the animal handling procedures were approved by the Institutional Review Board of Government College University Faisalabad, Pakistan (Ethical approval: GCUF/ERC/1972).

Acute Skin Irritation Study

An acute skin irritation study (14 days) was performed to evaluate the toxic and undesirable effects of Miet on skin. In brief, Miet was directly applied on the shaved area of skin of female mice (n = 5) for 14 days and observed daily for changes in skin color, any sign of inflammation or tissue injuries, and mortality. After 14 days, animals were euthanized and subjected to histopathological and hematological examinations of excised organs and blood samples.

Preparation of Stock Solutions

A tumor initiator stock solution (100×, 2500 nmol/0.2 mL acetone) of DMBA (7–12-dimethyl benz[a]anthracene) was prepared by dissolving 15 mg of DMBA in 4.68 mL of acetone under yellow or subdued light. 1× (25 nmol/0.2 mL of acetone) working solution was prepared by taking 150 μL from 100× and dissolving it in 14.85 mL of acetone. A stock solution (1% v/v) of croton oil was prepared by dissolving 1 μg in 100 μL of acetone.

Selection of Animals

Thirty female albino (nonpregnant and nulliparous) mice (n = 6), weight 25–30 g and 6–8 weeks old, were obtained from the National Institute of Health, Islamabad, and housed under controlled conditions of temperature (25 °C ± 2 °C) and humidity in the animal house of the Department of Pharmacology, Faculty of Pharmaceutical Sciences, Government College University Faisalabad, Punjab, Pakistan. The dorsal area of animals was shaved 3 days before commencement of experiment.

Experimental Design

Animals were divided in five groups of six mice each (Figure ) and were treated as

Figure 7

Methodology followed during study of antitumor activity.

Vehicle treated group: In this group all animals received acetone topically and double distilled water (100 μL/animal/day) orally continuously until the termination of study.

Carcinogen treated group: In the first week of the study, animals received a single dose of DMBA (25 nmol/0.2 mL of acetone) as a tumor initiator, and after 2 weeks of initiation, croton oil (1% w/v) was applied as a tumor promoter on the shaved area until week 16.

Miet 25% treated group: Animals received a single dose of DMBA in the first week, and after 2 weeks, Miet (25%) cream was applied topically. Thirty minutes after application of Miet 25% cream, croton oil was applied 3× per week on alternate days.

Miet 50% treated group: Animals received single dose of DMBA in the first week, and after 2 weeks, Miet (50%) cream was applied topically. After 30 min, croton oil was applied 3× per week on alternate days.

Standard (5-fluorouracil) treated group: Animals received a single dose of DMBA in the first week, and after 2 weeks, 5-FU 5% (5 mg/mL) cream was applied topically. Thirty minutes after application of 5-FU, croton oil was applied at 3× per week on alternate days.

During the whole period of 16 weeks, morphological parameters (tumor incidence, tumor yield, tumor burden, and cumulative number of tumors) were observed. At the end of the study, animals were sacrificed and subjected to hematological and histopathological examinations. Serum levels of carcinoembryonic antigen (CEA) and TNF-α were estimated using ELISA techniques.

Similarly, for the active constituent’s study (compound study), the experimental design was followed according to the Figure .

Figure 8

Methodology and steps involve in compound study.

Antitumor Activity of Active Compounds

Based on HPLC and previous GCMS[14] analyses of Miet, two compounds, i.e., coumarin and quercetin, in the same percentage as present in Miet were selected for detailed antitumor studies. In brief, coumarin and quercetin creams (36% and 0.76%) were applied topically following the protocol described in Figure .

In Vivo Antiangiogenic Activity

Chicken chorioallantoic membrane (CAM) assay was performed to evaluate the in vivo antiangiogenic activity of Miet following the reported protocols.[29] In brief, 30 leghorn chicken eggs (3–4 days fertilized) were divided into three groups as

Control group: Received same volume of ethanol loaded disk as used in Miet-loaded disk preparation.

Miet-treated groups: One group received 100 μg/mL of Miet and the second group received 200 μg/mL of Miet. After being sealed with paper tape, the eggs were placed in the incubator for 24 h. Later, eggs were opened to analyze the effects of Miet and control on the vasculature development.

Enzyme-Linked Immunosorbent Assay to Measure Serum TNF-α and CEA levels

Serum levels of TNF-α and carcinoembryonic antigen (CEA) were evaluated using ELISA kits following the manufacturers’ protocols.

Statistical Analysis

Data presented as mean ± SD (n = 6) of three independent experiments. One-way ANOVA followed by Post hoc Tuckey’s was performed to analyze the difference between different treatment groups.

Molecular Docking or In Silico Studies

In the current study, our tested bioactive chemotype coumarin (CUM) has been docked into the binding site of various proteins (CD31, TNF-α, VEGFR, BCL2, ROS1, and EGFR) to elucidate and compare the binding mode of CUM with the standard compound curcumin (CUR) bonded to same molecular targets. The 3D bioactive conformations of ligands CUR and CUM (Figure ) were built using SKETCH module in Sybyl-X 1.3,[30] followed by energy minimization according to Tripos force field with Gasteigere-Hückel atomic charge. The obtained energy optimized structures of CUR and CUM were used as initial conformers for molecular docking studies. Crystal structures of studied proteins CD31, TNF-α, VEGFR, BCL2, ROS1, and EGFR (5C14,[31] 5MU8,[32] 4AGD,[33] 4G3D,[34] 3ZBF,[35] and 1XKK,[36] respectively) were retrieved from the RCSB Protein Data Bank (http://www.rcsb.org). The obtained cocrystal complexes of proteins were further optimized prior to the docking studies using structure preparation tools included in biopolymer module of SYBYL-X 1.3.[30] Missing hydrogens were added; charges were applied, and atom types were assigned according to the AMBER 7 FF99 force field followed by energy minimization. The energy-optimized structures of selected proteins were carefully inspected to avoid any steric clashes and saved for further docking analysis.

Finally, the bioactive conformations of both ligands CUR and CUM were individually docked into the selected proteins using the Surflex dock module of Sybyl-X 1.3[30] by adopting the same protocol and parameters as reported in previous publications by Chohan et al.[37−39] The top 20 ensemble docking conformations ranked according to the cumulative score (CScore) function were saved for each ligand–protein complex. Only the ligand conformations with highest ranking poses, according to the CScore, were selected to investigate and compare key molecular interactions of the studied ligands in different proteins.