Phyto-compounds facilitated bio-fabrication of nano particles has received an unprecedented significance for the enhancement of bio-efficacies of allopathic drugs. This enthusiasm has motivated us to investigate Green Nanotechnological enhancement in the bio-efficacy of homeopathic drugs using gold nanoparticles embedded with biomolecules present in homeopathic mother tincture. The present study warrants the synthesis of gold nanoparticles using Carica papaya homeopathic mother tincture in a rapid single green process. Gold nanoparticles embedded with Carica papaya homeopathic mother tincture (CpAuNps) have been thoroughly characterized for their optical property, surface morphology, crystalline lattice, particle size, and zeta potential using UV-Vis spectroscopy, FE-SEM, XRD, and DLS and monitored for the enhancement in the platelets and Complete blood counts. Clinical trials (as per regulations laid down by Central Council of Homoeopathy, N. Delhi) have been performed on 30 patients (Age: 15-20 years) suffering from dengue fever, admitted in Faculty of Integrated Medicine Homoeopathic College & Hospital. Their blood samples have been collected at regular intervals under the supervision of the Medical Officer. Blood smears have been prepared at every alternate day to evaluate hematological parameters (platelet count) and CBC. The treatment of CpAuNps has depicted statistically significant enhancement (p<0.05) in platelet counts and effective in maintaining CBC in normal range. The enhancement in the platelets and maintenance of CBC may be ascribed to the activation of the gene expression of ALOX12 (arachidonate 12-lipoxygenase or platelet-type lipoxygenase) and PTAFR (platelet-activating factor receptor gene) enhancing the number of megakaryocytes. Such metabolic changes are likely due to the result of induced green nano technological modifications involving high surface area to volume ratio, biocompatibility and astonishing optical properties related to surface plasmon resonance. Overall, phenomenon allows accumulation and penetration of nano CpAuNps into the living tissues comparatively deeper.

Keywords: Homoeopathic mother tincture, Green synthesis, CpAuNps, Enhancement in platelets and CBC

INTRODUCTION

AuCl₄ + 3R-OH -------------> Au⁰ + 3R=O + 3H⁺ + 4Cl^-

Characterization of gold nanoparticle

Ultraviolet-visible spectrophotometer: The optimized experimental conditions of bio-fabrication of gold nanoparticles were as follows: CpMT (2 ml), sodium tetrachloroaurate dehydrate solution (3 ml; 1 mM) and sonication (45 min; 50 KHz) at pH 6.8. It was found that at 2:3 ratio of CpMT and NaAuCl₄.2H₂O solution had a maximum absorption band at 543 nm (Figure 1). Any further dilution didn’t show any marked increase in the absorbance, therefore, the ratio 2:3 was considered optimum.

X-ray diffraction: Bragg diffraction peaks 2θ appeared at 38.18°, 44.3°and 64.9° in the embedded gold nanoparticles (Figure 2). It could be indexed to (111), (200) and (220) having lattice planes of face-center cubic compared with (JCPDS file 65-2870). The intensity of the diffraction peaks (200) and (220) were found lower than the corresponding crystallographic plane (111). The fact established that the lattice plane (111) is the transcendent crystallographic plane and is more reactive because of its high atom density [21]. The observed noise and broadening of the peak in XRD record of Gold nanoparticle may be assigned to the existence of medicinally important secondary metabolites (flavonoids) loaded on the surface of bio-fabricated embedded gold nanoparticles.

FE-SEM: FE-SEM images (Figure 3) acquired from the drop-coated film of nanoparticles indicated polydispersed spherical shaped morphology of bio-fabricated gold nanoparticles. 

Dynamic light scattering: The dynamic light scattering (DLS) spectrum highlighted the asymmetric distribution of nanoparticles mainly in the range of 28 to 130 nm (Figure 4a). However, a little population was extended in the range of 1200 nm. The average hydrodynamic size (Z-Average) of embedded gold nanoparticles was found to be 62.8 nm. Zeta potential of embedded gold nanoparticles determined in water medium as a dispersant was -10.9 mV (Figure 4b). The magnitude of observed high negative charge on the bio-fabricated nanoparticles might be acting as a repulsive barrier, avoiding aggregation of nanoparticles.

Estimation of platelets count and CBC

A perusal of Table 1 depicts time-dependent (1^st-7^th day) increase in platelet count of normal control and CpMT and CpAuNps treated groups. However, in the normal control consisting of dengue patient group, there is an initial slight decrease in platelet count followed by a gradual increasing pattern but not to the level of platelet counts expected in a healthy person. Interestingly, about 50% enhancement in the platelet counts has been observed in the case of CpAuNps compared to CpMT treated groups. An increasing trend (1^st-7^th day) has been observed in the case of CBC in normal control reaching to the value not expected for a normal health (Table 2). The treatment of CpMT and more effectively of CpAuNps has been found quite effective for maintaining CBC to a level of normal range (6,000-10,000 cells/µL).

The observed enhancement in platelets and CBC may be assigned to the high surface area to volume ratio (nano sizing), biocompatibility, astonishing optical properties related to surface plasmon resonance and coating of medicinally important Carica papaya secondary metabolites (polyphenolic flavonoids) on freshly prepared nanoparticles. Overall, phenomenon allows accumulation and penetration of nano CpAuNps into living tissues comparatively deeper.

Phytochemical investigation on Carica papaya has reported the presence of various glycosidic flavonoids and terpenoids [22]. Polyphenolics like flavonoids and related compounds have been reported to enhance platelet and CBC in the patients of dengue fever [23,24]. It is reported that the extract of papaya enhances the gene expression of arachidonate 12-lipoxygenase or platelet-type lipoxygenase (ALOX12) and platelet-activating factor receptor gene (PTAFR) many folds finally promoting platelet production via the increased number of megakaryocytes [25].

CONCLUSION

The present study explores the role of green nanotechnological enhancement in the bio-efficacy of any homeopathic mother tincture. Gold nanoparticles are bio-fabricated using homeopathic Carica papaya mother tincture in a single step green method. After thorough characterization of bio-fabricated gold nanoparticles embedded with biomolecules of Carica papaya tincture (CpAuNps) was monitored for enhancement in platelet counts and CBC of patients suffering from dengue fever. The statistically significant enhancement in platelet and CBC of the patients was observed. The action of bio-fabricated gold nanoparticles (CpAuNps) at the nuclear level on the gene expression of arachidonate 12-lipoxygenase or platelet-type lipoxygenase (ALOX12) and platelet-activating factor receptor gene (PTAFR) is under study.

ACKNOWLEDGEMENT

The authors gratefully acknowledge Prof. P. K. Kalra, Director, and Prof. Sahab Dass, Head Department of Chemistry, Dayalbagh Educational Institute for providing necessary facilities. Authors also thank Dr. Vijai Kumar, Advisor, Medical and Health Practices, Dayalbagh Educational Institute for discussions.

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