ANTI-MELANOMA BIO-EFFICACY OF THE PLANT MADHUCA LONGIFOLIA AND ITS ENHANCEMENT USING BIOACTIVE PRINCIPLE LOADED GOLD NANOPARTICLE

Objective: Unexplored in-vivo anti-melanoma bio-efficacy of the plant Madhuca longifolia (bark) has been carried out against C57BL/6 mice. 
Methods: Optimized experimental conditions of phytofabrication of gold nanoparticles were as follows: flavonoid content (1 ml, 0.5 mg/ml), sodium tetrachloroaurate dihydrate solution (2 ml, 1 mM), and sonication (15 min, 20 KHz) at pH 4. The optical properties; ultraviolet-visible spectrophotometer (UV-Vis), particles size and zeta potential (Zetasizer), miller indices; X-ray diffraction (XRD), morphology; field emission-scanning electron microscope (FE-SEM), particle size; high resolution-transmission electron microscopy (HR-TEM), surface roughness; atomic force microscopy (AFM) and elemental composition; and energy dispersive X-rays (EDX) of flavonoid loaded gold nanoparticles. In-vivo anti-melanoma bio-efficacy has been carried out against C57BL mice. Radioisotopic, hematological, and histopathological studies were carried out using standard procedures. 
Results: Redox potential of the total flavonoid extracted from the bark of the plant (Madhuca longifolia) has been used for the fabrication of flavonoid loaded gold nanoparticles (F@AuNp) and confirmed for the first time their significant anti-melanoma bio-efficacy. The finding is supported by hematological and histopathological studies carried out in the organs (liver, kidney, and intestine) of C57BL mice. The significant enhancement in phytofabricated F@AuNp compared to native bark extract of the plant has been assigned to enhanced stay period and nanosizing, biocompatibility, nontoxic nature, and enhanced beneficial payload to the cancerous cells. 
Conclusion: Such phytofabricated gold nanoparticles possess an admirable prospect for the expansion of herbal nanomedicine for anti-melanoma bio-efficacy.


INTRODUCTION
Recent realization that folk plants indicated for various bio-efficacies should be established scientifically and to make the use of their active template molecules in biomedical research. Plant secondary metabolites such as vinblastine, vincristine, etoposide, teniposide, taxanes, camptothecin, irinotecan, and topotecan have been well recognized as antioxidants and anticancer bio-agents for direct inhibition of cell proliferation [1][2][3][4][5]. The meagre in-vivo bioavailability of plant secondary metabolites [6] owing to their rapid and extensive metabolism has directed research efforts toward the enhanced accumulation of phyto-modalities in the tumor cells with no or minimum potential toxicity to healthy cells [7,8]. Plant-mediated fabrications of noble metal nanoparticles have signified themselves as an important tool for the enhancement in the pharmacological efficacies. Research devotion has been paid toward the use of diverse plant extracts as such or their bioactive constituents for the phytofabrication of metal nanoparticles [9][10][11][12][13][14][15][16][17][18][19][20][21][22].
Madhuca longifolia (Mahua) belongs to the Sapotaceae family and is an Indian tropical tree found in most part of the country like West Bengal, Maharashtra, Madhya Pradesh, Kerala, Gujarat, Orissa, Chhattisgarh, Jharkhand Uttarakhand, and Uttar Pradesh [23][24][25]. Among the tribe's society, its different parts are used as folk remedies for skin-related issues, wound healing, swelling, antioxidant, and local liquor formation [26][27][28]. Phytochemical screening of various parts of the plant Madhuca longifolia has been restricted mainly to qualitative analysis, exploring the presence of polyphenolics, flavonoids, terpenoids, saponins, tannins alkaloids, glycosides, and large series of plant acids [29]. Quantitative analyses using gas chromatography-mass spectrometry (GC-MS) and high-performance thin-layer chromatography (HPTLC) have shown the presence of a considerable amount of polyphenolics type compounds, confirming the presence of quercetin as flavonoid [30]. However, the presence of a pentacyclic triterpene derivative in a small amount has also been elucidated on the basis of extensive spectroscopic study [31]. The plant lacks detailed quantitative analysis. Madhuca longifolia is a folk plant of our current interest aiming for its nanoscale pharmacological perspectives.
In the perpetuation of our work on phytofabrication of noble nanoparticles for the enhancement of various bio-efficacies [32][33][34][35][36][37], we have recently explored in-vitro anti-melanoma bio-efficacy of the plant Madhuca longifolia (bark) against mice (B16F10) and human (A375) melanoma cell lines [35]. The present communication for the first time reports in-vivo anti-melanoma bio-efficacy of the plant Madhuca longifolia (bark) against C57BL/6 mice model. The witnessed anti-melanoma bio-efficacy has been successfully enhanced using gold nanoparticles loaded with flavonoids. The proposed flavonoid loaded gold nanoparticles are labeled as (F@AuNp) and used throughout the manuscript. Such medicinally benign active principle loaded gold nanoparticles possess an admirable panorama for the advancement of commendation of herbal Nano-medicine.

Extraction and characterization of flavonoid
Microwave-assisted extraction of the plant (bark; 5 g) was carried out in aqueous-alcoholic solution at 200W for 5 min and was used for the quick test of the formation of gold nanoparticles. For a detailed study, cleaned, shade dried, and powdered bark (200 g) was defatted with petroleum ether (60-80°C) for 24 h. The defatted extract was subjected to the Soxhlet extraction with aqueous-ethanol for 72 h. The extract was concentrated by rotavapor distillation and finally dried by purging nitrogen and stored (bark extract=15.78 g) in the dark at ±4°C. Column chromatographic separation of the fraction (10 g) was carried out (length 120 cm; diameter 4 cm; mesh size 60; stationary phase; silica gel [125 g]) and washed with CH 3 OH: CHCl 3 (3:7). After the removal of the solvent, a brown mass was obtained. The brown mass was re-chromatographed using column (length 150 cm; diameter 3 cm; stationary phase; silica gel [80 g]) and eluted [35] with solvent mixture CHCl 3 : MeOH (8:1). The brown mass portion was subjected to high-performance liquid chromatography-electrospray ionizationquadrupole time-of-flight-mass spectrometer (HPLC-ESI-QTOF-MS) for the characterization of the flavonoid compounds. HPLC-ESI-QTOF-MS analysis was performed on a quadrupole time-of-flight (QTOF) mass spectrometer connected with Agilent 1200-HPLC system through dual electrospray ionization interface (Agilent Technologies, USA). HPLC separation was carried out on a Poroshell 120 EC-C18 column (50 mm×4.6 mm, 2.7 μ). The mobile phase consisted of 0.1% formic acid solution (A) and methanol (B) with a flow rate of 0.5 ml/min under the gradient program of 30-90% (B) for the initial 10 min, then 90% (B) from 10 to 20 min, 90 to 30% (B) from 20 to 25 min, and 30% (B) from 25 to 30 min. The HPLC-ESI-QTOF-MS was carried out in positive ESI mode. The resolving power of the QTOF analyzer was set above 10,000 (FWHM). The spectra were acquired within a mass range of m/z 100-1500. The capillary temperature was set to 350°C, N 2 nebulizer pressure (40 psi), and gas flow rate (10 l/min).

Animal experiments
Random breed, 5-6 weeks old, and 24-28 g body weight (bw) bearing C57BL healthy mice were maintained under controlled standard environmental conditions of the temperature at 25±1°C with relative humidity (55-65%) under dark and light cycle (14 h:10 h). The animals were fed with standard pellet diet and tap water ad libitum.

Ethics statement
The Guidelines of the Committee (CPCSEA) was followed for all the animal experiments against ethical permission No. 1698/PO/a/13/ CPCSEA, Govt. of India.

Experimental designing
Acute oral toxicity test was performed as per the Organization for Economic Co-operation and Development (OECD) guideline 423 (OECD, 2010) on C57BL/6 mice. The animals were randomly divided into six groups of six animals each. Cells (2×10 5 /ml) in phosphate buffer saline and suspension (200 μl) were maintained and subcutaneously injected into mice on the dorsal side. After the 4 th day of injection, the tumor started budding. At the palpable stage of the tumor, the treatments at (mg/kg, bw) of mice were given orally. Cyclophosphamide (reference drug; 150 mg/kg) was intraperitoneally injected every day for 21 days. During the treatment, the size of the implanted tumor was measured at regular time intervals. Group, I served as normal control (NC). The remaining animals were inoculated with B16F10 (cells/mice) and further divided into five groups. Group II served as tumor control (TC), while Group III served as a positive control (PC) and treated with the reference drug (cyclophosphamide; 150 mg/kg; bw). Group IV (BE) was treated with aqueous ethanolic bark extract (500 mg/kg; bw). Group V (FC) was treated with flavonoid content (250 mg/kg; bw). Group VI was treated with F@AuNp (150 mg/kg; bw). All the treatments were given orally after 24 h of inoculation and continued for 21 st days (once daily). After the last dose, all mice from each group were sacrificed. All the treatments were administered orally through a metal oropharyngeal cannula while the reference drug was given intraperitoneally. Cyclophosphamide, an anti-neoplastic drug frequently used in treating malignancies; therefore, it was used in the present study.
To observe any life-threatening toxicity of the samples and reference drug, body weights of all animals were measured daily during the treatment period. Tumor growth was determined by measuring the diameter using digital Vernier Caliper daily from the very 1 st day of the treatment. Tumor volume (in mm 3 ) was calculated [38]. Tumor growth delay was determined by the standard formula: T-C, where T represents median time (in days) required for the tumor's to reach a volume of 100 mm 3 and C represents median time (in days) required for the control group to reach the same size [39].

Hematological parameters
The measurement of haematological parameters [haemoglobin, red blood cells, white blood cells, neutrophils, lymphocytes, and platelets counts] were carried out on the first day in blood sample (0.3 ml) taken from retro-orbital plexus in ethylenediaminetetraacetic acid (EDTA) tube. After 24 h of the tumors cell's inoculation, the animals were given as per designed group treatments (once daily for the next 20 days). On the 21 st day, animals were sacrificed and the assessments of haematological parameters were again conducted.

Radioisotopic study
All the treatments were tagged with radiolabelled Tc 99 using stannous chloride reduction [40]. The stannous chloride (SnCl 2 ·2H 2 O) (1 mg/ml; 0.1N HCl) and F@AuNp were mixed in a vial containing (100 μCi;3.7 MBq 99 TcO 4-) procured from the Department of Nuclear Medicine, Jawaharlal Nehru Cancer Hospital and Research Center, Bhopal, India. The reaction mixture was vortexed and kept at ambient temperature. All the treatments tagged with Tc 99 were administered in mice through the tail vein and scanned using the Brivo NM 615 scanner at different time intervals.

Histopathological studies
The body organs (liver, kidneys, and intestine) were removed and processed overnight for dehydration, cleaning, impregnation in an automatic tissue processor (Sakura, Japan) and fixed in 10% the solution of neutral buffered formalin. The organs were embedded in paraffin blocks followed by sectioning (sections of 5 μm) and staining with hematoxylin and eosin. Slides were examined under a light microscope. The alterations compared to the normal structure were registered. The tumors from each group were dissected and subjected to the abovementioned treatment course and stained using hematoxylin and eosin. The architecture of the sections was examined using a microscope [41].

Statistical analysis
One-way ANOVA (Tukey-Kramer) was used for statistical analysis and results were given as mean±SD. The significance of the value was considered at p<0.05 and p<0.01.

RESULTS AND DISCUSSION
Among the plant secondary metabolites, flavonoids are a broad class of polyphenolics biomolecules with numerous hydroxyl groups and are capable to interact with cell membranes and penetrate more or less deep into hydrophobic cellular sites. They have high medicinal potential behaving as strong antioxidants. Antioxidants, as immunity boosters, have also been proved to strengthen the anticancer activity [42][43][44]. Relevant literature indicates the presence of flavonoid in a substantial amount in the plant Madhuca longifolia. The total phenolics and flavonoids content in aqueous ethanolic extract of the bark was experimentally measured and found in quite a substantial amount (35.94±0.15 mg/g) and (12.15±0.15 mg/g), respectively. The fact has motivated us to quantify the presence of total polyphenolics and flavonoids in the target plant and to study their role toward in-vivo anti-melanoma bio-efficacy.
The detailed LC-MS characterization of flavonoids extracted from the plant (bark), fabrication of gold nanoparticles loaded with total flavonoid content, and their characterization have been published in our earlier publication [35] in which we have also demonstrated noncytotoxicity of bark extract and flavonoid loaded gold nanoparticles (F@AuNp) toward the normal lymphocytes cells at the doses selected for the in-vitro assessment of anti-melanoma bio-efficacy. However, the relevant information has been summarized as follows. HPLC-ESI-QTOF chromatogram (Fig. 1)  The strong synergistic reduction potential of a family of flavonoids extracted from the bark of the plant M. longifolia was used for the phytofabrication of F@AuNp. Table 1 presents the complete characterization of phytofabricated F@AuNp at a glance.

Enhancement of stay period
To monitor the stay period of extracted flavonoid and its loaded gold nanoparticles, radiolabelling experiments were conducted using Tc 99 as a radioactive tracer having photon emission energy (140 KeV) and half-life (6.0058 h). Radioisotopic scintigraphic images of the melanoma tumors exhibit 4 and 6-h stay periods of flavonoid content and radiolabelled F@AuNp, respectively (Fig. 2). After 4 h, native flavonoid content starts dissipating from the target site. The short-stay period of native phytoextract has also been noticed earlier [44,45]. The present study demonstrates an improved stay period of gold nanoparticles loaded with plant flavonoids, finally leading to improved bio-availability.

Noncytotoxic potential of phytotreatments against human lymphocytes cells
The mean body and tumor weights, tumor volume, and delay in tumor growth (21 st days) in C57BL/6, mice model of all the experimental group's negative control (NC), tumor control (TC), bark extract (BE), flavonoids content (FC), flavonoids loaded gold nanoparticles (F@ AuNp), and positive control (PC) have been tabulated ( Table 2). All the experimental groups were also periodically monitored for any non-specific toxicity such as food and water withdrawal with impaired movement.
A perusal of Table 2     Each value is mean±SD (n=6), significance at (p<0.05) Normochromic cells, intact serosa and muscularis, with inflammation in crypt and villi. Group VI (F@AuNp): A large number of healthy crypt and villi, proper shape of serosa, and muscularis layers. It is inferred that there are no marked changes in the normal architectures (NC) of the target organ treated with F@AuNp. However, the reference drug (cyclophosphamide) treated group (PC) demonstrates noticeable damage to the normal architecture of body organs, warning the specific toxic nature of the reference drug. Other chemotherapeutic anticancer agents have also been reported to induce specific toxicity [46,47]. The C57BL/6 mice tumors of all the experimental groups were also subjected to histopathological studies (Fig. 6 The above observations demonstrate that among all the phytotreatments, there is a progressive increase in the anti-melanoma bio-efficacy in terms of apoptosis and necrosis, progressing from BE to FC and reaching to the optimum level in F@AuNp. The significant inhibition in angiogenesis and micro-vessels in F@AuNp treatment is the sign of its accumulation in tumor cells. The suppressed angiogenic vascularization has been reported to lead inhibition of tumor cell proliferation and apoptosis [48,49]. Finally, observations on hematological parameters of all the experimental groups of C57BL/6 mice also provide support to the    (Table 3).
The observed increase in WBC count in phytotreatments is a sign of development of a defense mechanism against malignancy which is in the divergence of the antitumor effect of a chemotherapeutic agent exhibiting a decrease in the WBC count [50]. All the phytotreatments again appear to reduce HB and RBC to a lesser extent compared to the reference drug. The development of anemia, an issue with most of the cancer chemotherapeutics and is faced as chemotherapeutic agentinduced myelosuppression activity [51]. The perceived increase in neutrophils and platelet counts and a decrease in lymphocytes in all the tumor-bearing animals relate with the promotion the proliferation of cancerous cells [52]. Thus, biocompatibility, tunable optical and electronic properties, and capping of medicinally important secondary metabolites (flavonoid) designate proposed nanoparticles (F@ AuNp) a suitable phytoagent to be developed for an upcoming drug Yadav et al.
delivery system. Nanosizing, inert character, and capability to attach with multiple surface components permit such plant composite nanoparticles to go through inside the cells and transport their payloads without eliciting an important immune reaction and any specific toxicity.
The exact mechanism of anti-melanoma action and its enhancement through F@AuNp has not been fully elucidated. Based on our experimental findings and pertinent information available, a tentative mechanism has been proposed in a précised schematic fashion (Scheme-1).

CONCLUSION
The native extract of the various parts of the folk plant M. longifolia has been studied for various bio-efficacies. However, it lacks phytochemical screening and characterization of bioactive principles. The present communication for the first time reports its unexplored anti-melanoma bio-efficacy of native bark extract, extracted total flavonoid (possible bioactive principle characterized using HPLC-ESI-QTOF-MS), and flavonoid loaded gold nanoparticles (nanoscale perspective) against C57BL/6 mice. The proposed phytofabricated gold nanoparticles with no sign of any specific toxicity toward human lymphocytes cells with enhanced anti-melanoma bioefficacy may be proved superior compared to the reference drug (cyclophosphamide).