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ORIGINAL RESEARCH

New Approaches in Breast Cancer Therapy Through

Green Nanotechnology and Nano-Ayurvedic
Medicine – Pre-Clinical and Pilot Human Clinical
Investigations
This article was published in the following Dove Press journal:
International Journal of Nanomedicine

Menka Khoobchandani,1,2 Kavita K Katti,1,2 Purpose: The overarching objective of this investigation was to investigate the intervention
Alice Raphael Karikachery,1,2 of green nanotechnology to transform the ancient holistic Ayurvedic medicine scientifically
Velaphi C Thipe, 1,2 Deepak Srisrimal,3
credible through reproducible formulations and rigorous pre-clinical/clinical evaluations.
Darsha Kumar
Dhurvas Mohandoss,3 Methods: We provide, herein, full details: (i) on the discovery and full characterization of
Rashmi Dhurvas Darshakumar,3 gold nanoparticles-based Nano Swarna Bhasma (henceforth referred to as NSB drug); (ii) In
Chintamani M Joshi,3 Kattesh V Katti1,2,4 vitro anti-tumor properties of NSB drug in breast tumor cells; (iii) pre-clinical therapeutic
1
Department of Radiology, University of efficacy studies of NSB drug in breast tumor bearing SCID mice through oral delivery
Missouri, Columbia, MO 65212, USA; 2Institute protocols and (iv) first results of clinical translation, from mice to human breast cancer
of Green Nanotechnology, University of
Missouri, Columbia, MO 65212, USA; patients, through pilot human clinical trials, conducted according to the Ayurveda, Yoga and
3
Dhanvantari Nano Ayushadi Pvt Ltd, Chennai Naturopathy, Unani, Siddha and Homoeopathy (abbreviated as AYUSH) regulatory guide-
600017, India; 4Department of Physics,
Department of Pharmacology, Department of lines of the Government of India in metastatic breast cancer patients.
Biological Engineering, University of Missouri Results: The preclinical in vitro and in vivo investigations, in breast tumor bearing mice,
Research Reactor (MURR), University of
Missouri, Columbia, MO 65212, USA established unequivocally that the NSB Nano-Ayurvedic medicine-gold nanoparticles-based
drug is highly effective in controlling the growth of breast tumors in a dose dependent fashion
in vivo. These encouraging pre-clinical results prompted us to seek permission from the Indian
Video abstract Government’s holistic medicine approval authority, AYUSH, for conducting clinical trials in
human patients. Patients treated with the NSB drug capsules along with the “standard of care
treatment” (Arm B) exhibited 100% clinical benefits when compared to patients in the treatment
Arm A, thus indicating the tremendous clinical benefits of NSB drug in adjuvant therapy.
Conclusion: We have succeeded in clinically translating, from mice to humans, in using
proprietary combinations of gold nanoparticles and phytochemicals to develop the Nano-
Ayurvedic drug: Nano Swarna Bhasma (NSB), through innovative green nanotechnology, for
treating human metastatic breast cancer patients.
Keywords: gold nanoparticles, mangiferin, mango peel, Nano Swarna Bhasma, NSB, triple
negative breast tumor, pilot clinical

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Introduction
The traditional Indian holistic medicine (Ayurveda), with over 5000 years of rich
Correspondence: Kattesh V Katti history, is gaining considerable acceptance in recent years because plant-based
Department of Radiology, University of
Missouri, One Hospital Drive, Columbia, preparations, which play a major role in the Ayurvedic healing processes, have
MO 65212 USA shown reliable curative effects in many chronic conditions.1–10 Approximately
Tel +1 573 882-5656
Email [email protected] two-thirds populations of the developed and developing countries have reported

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using one or the other form of alternative or complemen- world.52,53 In order to circumvent this long-standing pro-
tary medicine.11–15 Extensive literature on Ayurvedic blem, we have developed an innovative green nanotechnol-
medicine supports claims of significant improvement in ogy approach for the production of gold nanoparticles
patients’ symptoms thereby enhancing their quality of (AuNPs) employing complete scientific control on its for-
life.16–18 Despite the long history of Ayurvedic medical mulation for reproducible large-scale production as shown
modality, rigorously tested and scientifically reproducible in Figures 1 and 2. Our overarching rationale was to
formulations of Ayurvedic-relevant polyherbal drugs develop reproducible formulations of Swarna Bhasma,
for the treatment of various diseases and disorders through green nanotechnology, using various phytochem-
remain largely unestablished. The lack of reproducible icals adhering to strict Ayurvedic principles. We have
Ayurvedic formulations have also impeded the develop- coined a new term referred to as “Nano-Ayurvedic
ment of reliable clinical trials data from human patient Medicine” in order to capitalize on the important role of
populations. Overall, a myriad of scientific reasons have nanotechnology in making Ayurvedic medicine more effec-
contributed to significantly limited acceptability of tive. The term “Nano-Ayurvedic medicine” has been
Ayurvedic medicine within the global patient population. recently approved by the US Patents and Trade Marks
As part of our continued quest to develop scientific ratio- office.54 We have succeeded in using proprietary combina-
nale for Ayurvedic medicine, we have been researching, tions of gold nanoparticles and phytochemicals from
for over two decades, on the application of green nano- mango, turmeric, gooseberry and gum arabic to develop
technology to develop universally acceptable Ayurvedic Nano Swarna Bhasma through green nanotechnology. In
formulations.19–26 Green nanotechnology allows the pro- this report, we provide full details: (i) on the discovery
duction and efficient encapsulation of Ayurvedic-relevant and full characterization of gold nanoparticles-based Nano
phytochemicals on gold nanoparticles. Our long-standing Swarna Bhasma (NSB drug); (ii) Drug loading concepts to
scientific efforts, with a focus on gold nanoparticles, are prove that the gold nanoparticles produced through cock-
based on the rationale that in Ayurvedic medicine, gold tails of phytochemicals from mango peel contain predomi-
metal is ubiquitously used in combination with phyto- nantly the mangiferin functionalized gold nanoparticles; (iii)
chemicals from various herbs including cinnamon, In vitro anti-tumor properties of NSB drug; (iv) pre-clinical
clove, gooseberry, grape, mango, turmeric and a host of therapeutic efficacy of NSB drug in breast tumor bearing
herbs.19–26 Our efforts, serendipitously, led to a series of SCID mice through oral delivery protocols and (v) first
discoveries confirming that the phytochemicals used in results of pilot human clinical trials, conducted according
Ayurvedic medicines are indeed electron–rich antioxi- to the AYUSH regulatory guidelines in metastatic breast
dants and that the interaction of gold metallic precursors cancer patients. The primary objective of this pilot clinical
with phytochemically-harnessed electrons produced herb/ trials investigation was to assess the safety profile, and to
phytochemicals-encapsulated, well-defined, gold nano- determine the number of patients who experience adverse
particles. The large surface area of gold nanoparticles events by NCI toxicity criteria at every course. The second-
embedded with atomically active nano surface motif ary objective was to determine the efficacy of NSB drug
allowed highly efficient encapsulation of phytochemical-
(Trade name: DNANOSTANNA) in terms of response rate
(s) – thus paving, for the first time, an innovative green
in participating breast cancer patients assessed by radio-
nanotechnology route providing reproducible and scienti-
graphic evaluation by RECIST Criteria. Our overall assess-
fically verifiable Nano-Ayurvedic formulations.19–24,26–49
ments included evaluating the quality of life of patients who
Choice of gold nanoparticles is further necessitated by
participated in this study. The overall oncological implica-
the fact that this metal embodies the iconic Ayurvedic
tions of green nanotechnology-based Ayurvedic drugs are
medication “Swarna Bhasma” (SB) (“Swarna” meaning
discussed.
gold, “Bhasma” meaning ash) which contains gold particles
along with various herbal mixtures. Swarna Bhasma is an
ancient Indian medicine with established anticancer Materials and Methods
activity.50,51 Extensive studies have reported on the unchar- Preclinical Studies
acterized chemical features of SB thus making it difficult Materials
for clinical acceptance and penetration of this Ayurvedic Gold salt, and MTT assay kit (3-(4,5-dimethylthiazol-2-yl)-
drug within the larger patient populations across the 2,5-diphenyltetrazolium bromide) were obtained from Sigma

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Mangiferin from Mango peel

Anoxidant.….sensizer….
HO OH
HO OH
HO OH HO OH
HO OH HO OH HO OH
AuHO OH HO OH
HO OH
HO OH HO OH HO OH
HO OH AuHO OH
OH HO OH
NaAuCl4 HO OH
HO
HO OH
HO OH
HO
HO
OH
OH
HO OH HO OH
Gold salt HO OH HO OH HO OH
AuHO OH HO OH
HO OH
HO OH HO OH HO OH
HO OH AuHO OH
HO OH HO OH
HO OH
HO OH
HO OH

Mangiferin
Figure 1 Production of therapeutic MP-AuNPs through Green Nanotechnology.

+ NaAuCl4

Gold salt
Mangiferin

MGF-AuNPs
Figure 2 Production of therapeutic MGF-AuNPs through Green Nanotechnology.

(St. Louis, MO, USA). RPMI, fetal calf serum, TryplE, lines were obtained from the American Type Culture
Trypan blue and DAPI (4ʹ,6-diamidino-2-phenylindole) Collection (ATCC; Manassas, VA), and cultured by the
were obtained from Thermo Fisher Scientific, USA. Double University of Missouri Cell and Immunobiology Core
distilled water was used throughout the experiment. facility using procedures recommended by ATCC.
Mangiferin was purchased as a 99% pure phytochemical
from Sigma Aldrich and used without further purification. Characterization of Nano Swarna Bhasma (NSB)
Drug
Cell Line Transmission electron microscope (TEM; JEOL 1400, LTE,
The MDA-MB-231 (breast tumor cells from human breast Tokyo, Japan), Scanning Transmission electron microscope
tumor origin) and HAECs (human aortic endothelial) cell (STEM) and Electron Energy Loss Spectroscopy (EELS)

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images were used to characterize various gold nanoparticles. AuNPs were centrifuged twice in 2 mL eppendorf tubes at
Zeta potential was obtained by Zetasizer Nano S90 (Malvern 12,000 rpm at 12°C for 15 min to remove any unreacted
Instruments Ltd. USA) to evaluate the overall in vitro stabi- MGF or gold salt.
lity of nanoparticles against agglomeration. Gold nanoparticles obtained through mango peel
extracts (MP-AuNPs) as well as those produced through
NSB Drug Formulation the interaction with mangiferin (MGF-AuNPs) were char-
The main nanoparticulate drug in our formulation is acterized by UV-Visible absorption spectrophotometry, par-
derived from gold nanoparticles produced through the ticle size analyzer, and transmission electron microscopy
interaction of phytochemicals in mango peel with gold (TEM). The amount of Au was calculated by atomic
salt as discussed below: absorption spectrometry (AAS) technique. Nanoparticles
were stored at 4°C for further use in biological evaluations.
Preparation of Mango Peel Powder In order to adhere with the Indian traditional medicine
Mango (Mangifera indica) peel purchased from a reliable Ayurveda modality, we introduced proprietary combinations
herbal source from India was used in all the experiments. of additional plant phytochemicals from Amalaki (Emblica
Mango peel was washed with doubly ionized water to officinalis), Amra (Mangifera indica), Haridra (Curcumin
remove any contaminants or dust particles and incubated longa), Babbula (Acacia nilotica), Yashtimadhu (Glycyrrhiza
at 50°C for 4 hr and then grinded to obtain dry powder. glabra) with MP-AuNPs (Scheme 1). Overall, we call this
Mango peel powder was stored at room temperature and combination of MP-AuNPs with the proprietary phytochem-
used for subsequent gold nanoparticles synthesis. ical excipients as Nano Swarna Bhasma drug with the trade
name as DNANOSTANNA. NSB’s formulation uses unique
Synthesis of Mango Peel Phytochemicals Coated Gold green nanotechnology without the use of any toxic organic
Nanoparticles (MP-AuNPs) chemicals or solvents. All formulations are reproducible. NSB
After extensive experimentation, we arrived at the follow- was subjected to extensive in vitro and in vivo investigations
ing reproducible ratios of mango peel powder with respect using triple-negative breast tumor cells. The anti-tumor assays
to gold salt concentrations. Thirty milligrams of the dry are described below.
mango peel powder was added to 6 mL of DI water in
a 20 mL vial and stirred for 10 min at rt for the complete Cell Viability Assay
homogenous suspension. Then, 100 μL of 0.1 M NaAuCl4 The effect of NSB drug on breast cancer (MDA-MB-231)
solution was added and the color of the solution turned to and normal (HAECs) cells, viability was determined using
ruby-red within 2 min, indicating the formation of gold MTT assay. The intensity of developed color was mea-
nanoparticles. The solution was filtered to remove the sured by micro plate reader (Molecular device, USA)
remaining insoluble mango peel powder and used for full operating at 570 nm wavelength.55 Percent cell viability
characterizations as outlined below. was calculated by using the formula: (T/C) × 100, where
C = Absorbance of control, T = Absorbance of treatment.
Synthesis of Mangiferin (Major Phytochemical in Mango The IC-50 values were calculated using the Origin
Peel) Conjugated Gold Nanoparticles (MGF-AuNPs) software.
After extensive experimentation, we arrived at the following
reproducible ratios of mangiferin with respect to gold salt Animal Studies
concentrations. The mangiferin gold nanoparticles were pro- All experiments involving animals were approved by the
duced by mixing 4.2 mg of mangiferin (MGF) in 6 mL Institutional Animal Care and Use Committees (IACUC)
of doubly deionized (DI) water. The solution was stirred at of the Harry S. Truman Memorial Veterans Hospital and
100°C for 10 min to dissolve all the MGF in water to get the University of Missouri, and were performed according
a clear solution. Gold salt (100 µL of 0.1 M) was added to the to the Guidelines for the Care and Use of Laboratory
reaction mixture when a ruby-red coloration was observed Animals. Imprinting control regions-severe combined
within a few seconds of addition. The reaction mixture was immunodeficient (ICR-SCID) female mice (from Taconic
further stirred for an additional 2 hrs to make sure that all the Farms, Hudson, New York) were used for the therapeutic
gold was transformed to the corresponding mangiferin func- study. The mice used in our investigations weighed
tionalized gold nanoparticles (MGF-AuNPs). Finally, the 24–30 g.

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Emblica officinalis Glacirrhiza glabra

Mangifera indica
Curcumin longa Acacia niloca

NaAuCl4

NSB Drug
MP AuNPs
Extract
MP/MGF
funconalized AuNPs TEM image of MP-AuNPs
Producon of NSB with MP/MGF
funconalized AuNPs

Scheme 1 Gold nanoparticle phytochemicals incorporated 'Nano Swarna Bhasma' (NSB) formulation using proprietary amounts of phytochemicals from Amalaki (Emblica
officinalis), Amra (Mangifera indica), Haridra (Curcumin longa), Babbula (Acacia nilotica), and Yashtimadhu (Glycyrrhiza glabra).

The SCID female mice were subcutaneously inoculated Pilot Clinical Trial Investigations
with 10x106 MDA-MB-231 cells (suspended in 0.1 mL of After obtaining highly encouraging in vitro antitumor
sterile DPBS (Dulbecco’s phosphate-buffered saline) and assays and in vivo therapeutic efficacy studies in breast
Matrigel® (2:1, v:v)) in the right hind flank under inhalation tumor bearing mice, we obtained regulatory permission
anesthesia (isoflurane/oxygen). After inoculation, tumors were from the Indian government regulatory authority of
allowed to grow for 3 weeks, during this time tumors were AYUSH (DNA_SPN_B001_17) for initiating a pilot clin-
measured by digital caliper measurements and calculated as ical trial investigation in breast cancer patients. The details
length x width x height. The mice were randomly divided into of clinical trials planning, inclusion/exclusion criteria,
three groups (n=7/group) with no significant difference in dosage, endpoint and overall clinical efficacy results are
tumor volume, the day of randomization was considered summarized in the following sections:
as day zero of the therapy study. On day zero, mice were Here we describe the overall methodology of our study
treated orally with drug mixed in peanut butter as follows: aimed at evaluating the safety and efficacy of NSB drug
(herbal formulation) as a treatment for breast cancer. The
● Group 1- Untreated control (peanut butter) study followed a randomized, multicenter, parallel design in
● Group 2- NSB drug treated (3 mg drug/30 gm mouse); female patients with pathologically confirmed invasive
● Group 3- NSB drug treated (7 mg drug/30 gm mouse); breast carcinoma stage IIIA or IIIB. At visit 1, after the
study personnel explained the study procedures, written
Animals were treated orally twice per week until the end informed consent was obtained in the presence of the inves-
of the study. The animals were monitored for their tumor tigator from patients who were willing to participate in the
volume, body weight and overall health condition during the study. Each patient, then underwent screening procedures.
study period. At the end of the study, animals were sacrificed. Screening procedures included medical history, clinical
A separate group (n=7) was kept as a control group (no examination and laboratory investigations (hematology, bio-
tumor and no treatment). This group of animals served as chemistry, urinalysis and serology). Urine pregnancy test was
a control group for body weight measurements. Animals not performed as none of the enrolled female patients were of
were sacrificed at the end of the study. child bearing age. Patient’s electrocardiogram (ECG) and
X-ray were taken at the time of screening.
Statistical Analysis Each patient was screened for the presence of Breast
All experimental data for pre-clinical study were given as Carcinoma Stage IIIA or IIIB by computed tomography
mean±SEM. Statistical analysis was carried out using the (CT) scan (RECIST criteria) before enrolling into the
one-way analysis of variances (ANOVA) using Graph Pad study. Upon satisfying the inclusion and exclusion cri-
Prism software. P<0.05 was considered significant. teria as per protocol, patients were enrolled in the study.

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Inclusion and Exclusion Criteria ● Participants taking alternative therapies for cancer
Inclusion Criteria: Participants Who Met the Following must stop taking these therapies prior to randomiza-
Criteria Were Included in This Study tion. Alternative therapies were not allowed during
the treatment or follow-up portions of the study.
● Female patients between age 18 to 65 years of age ● Peripheral neuropathy ≥ Grade 2.
with pathologically confirmed invasive breast carci- ● ECG with significant abnormalities (as determined
noma stage IIIA or IIIB by the investigator)
● HER-2/Neu negative status or HER-2/Neu positive ● Participants who are medically unstable, including
status. but not limited to active infection, acute hepatitis,
● Bi-dimensionally measurable and evaluable disease gastrointestinal bleeding, uncontrolled cardiac
lesion. arrhythmias, interstitial lung disease, inflammatory
● No evidence of disease outside the breast or chest bowel disease, uncontrolled angina, uncontrolled
wall, except for ipsilateral axillary lymph nodes hypercalcemia, uncompensated congestive heart fail-
● Subject willing to participate by providing written ure, uncontrolled diabetes, dementia, seizures, super-
informed consent ior vena cava syndrome.
● Life expectancy must be ≥6 months
● Eastern Cooperative Oncology Group (ECOG) per- Our pilot clinical trials were based on a comparative
formance status with 0 or 1 study model with two arms. As per plan, a total of six
● Chest CT scan without evidence of metastasis at the patients enrolled in the study were randomized to either of
time of screening the two treatment arms: Treatment A and Treatment B in
1:1 ratio. As per the sponsor’s requirement, one additional
Laboratory value criteria was as follows: patient was enrolled later in the study to replace one with-
drawn patient from treatment arm B. Details of treatment
● White Blood Cells ≥3000/mm3 arm A and B are outlined in the following sections-
● Neutrophils ≥2000/mm3 All the eligible patients were informed by the site
● Hemoglobin ≥8 g/dL coordinator to visit the site for randomization within 14
● Platelets ≥100,000/mm3 days (± 7 days) of screening (visit 2) for baseline evalua-
● Serum creatinine ≤1.5 × Upper normal limit (UNL) tions. After randomization and all baseline measurements,
● Alkaline phosphatase ≤1.5 × UNL all eligible patients received their respective study treat-
● Total serum bilirubin ≤1.5 ×UNL for the institution. ments in the following manner:
● Serum Glutamic-Oxaloacetic Transaminase (SGOT)
≤1.5 × UNL Patients in Treatment Arm A Received the Following
● Serum calcium ≤ UNL “Standard of Care Treatment”
● Pregnancy test must be negative. Doxorubicin injection, concentration of 60 mg/m2, every 3
weeks as a 1 hr intravenous infusion. In addition, this
Additional criteria included that subjects with child bear- patient group also received Cyclophosphamide injection,
ing potential must be willing to agree to use effective concentration of 600 mg/m2, every 3 weeks as a 1 hr
contraceptive methods while on treatment and for 3 intravenous infusion.
months after the completion of study.
Patients in Treatment Arm B Received the Following
Exclusion Criteria Doxorubicin injection, concentration of 60 mg/m2, every 3
weeks as a 1 hr intravenous infusion, Cyclophosphamide
● Prior systemic treatment for breast cancer with hormonal injection, concentration of 600 mg/m2, every 3 weeks as
therapy, chemotherapy, or any other anticancer therapy. a 1 hr intravenous infusion. In addition, this group of
● Prior radiation therapy. patients were administered with the experimental NSB
● Participants who received or receiving or scheduled Drug in doses of two capsules thrice a day after food.
to receive post-operative radiotherapy. These doses for every patient was calculated based on
● History of a malignancy other than breast cancer body surface area (BSA). Four such cycles were repeated

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over a period of 12 weeks. Investigator dispensed suffi- Secondary Endpoint (Efficacy; RECIST Criteria)
cient quantity of NSB drug and patient diary to each Efficacy of the NSB drug in terms of response rate in partici-
patient was randomized in treatment B with dosing pating breast cancer patients were assessed by Radiographic
instructions. In the arm B, patients were instructed to Evaluation by RECIST Criteria. Improvement in Quality of
take two NSB drug capsules thrice a day after food with life comparing baseline with end of study was also used as
sufficient quantity of water by themselves at home. All a criterion for efficacy.
patients maintained records of each dosing details in the
patient diary. Details of dosing were documented appro- Statistical Analysis Related to Pilot Clinical Trial Study
priately in chronic renal failure (CRF) through patient’s As per the plan, the data from both the centers that partici-
physical examinations which included observation of vital pated in this protocol was used, so that an adequate number
signs. Laboratory investigations, including hematology, of patients were available for analysis. The data were ana-
clinical chemistry and urinalysis were done (except for lyzed by 15 Clinical Research Pvt. Ltd – a clinical trials
two patients −02-002 and 02–003 whose urinalysis was company based in Chennai, India. Efficacy variables were
not done for visit 1 and 3, respectively). Serology was analyzed using data from all patients to whom study treat-
performed at visit 1 only and was not performed at all the ment was assigned. Following the intent-to-treat principle,
visits as mentioned in the protocol. Patients were evalu- patients were analyzed according to the treatment they were
ated through quality of life (QoL) questionnaires at every assigned at randomization. As per plan, all efficacy variables
were analyzed by FAS and PPS. PP population consisted of
visit except visit 1. CT scan (RECIST criteria check) was
all those full analyses set subjects and those completed the
repeated at visit 4 and end of study visit post 12 weeks.
study without any major protocol deviation. None of the
Adherence to assigned regimen was assessed by recording
patients showed any major protocol deviation. Comparison
the number of capsules dispensed and returned by the
between the groups was performed for standard of care
patient at each visit. Study drug compliance was checked
treatment alone and standard of care treatment with NSB
by counting the quantity of returned/unused investigational
drug. All categorical variables were summarized in tabular
products and the review of the patient diaries, which they
form by means of frequency counts and percentages. All
brought at every visit. Unused amounts were documented
continuous variables were summarized in tabular form by
in the drug log book. Patient’s concomitant medication
presenting the n, mean, median, standard deviation (SD),
received was also documented. Adverse events were
minimum, and maximum.
assessed by NCI toxicity criteria at every course.
Safety
Number of Patients Planned and Analyzed Safety population comprised of all randomized patients who
Six patients were planned; seven eligible patients with received at least one dose of study treatment. Patients were
stage III A or III B breast cancer were enrolled; six analyzed according to the treatment received. Adverse
patients completed the clinical investigation. Out of events, vital signs, hematology, biochemistry parameters
seven patients enrolled into the study, one patient was were summarized descriptively by treatment groups.
withdrawn after visit 2 due to non-compliance. Full ana-
lysis set (FAS) and per protocol set (PPS) consisted of six Results for Preclinical Studies
patients.
Synthesis and Characterization of
MP-AuNPs and MGF-AuNPs
Efficacy and Safety Mangiferin (1,3,6,7-tetrahydroxyxanthone-C2-D glucoside;
As part of efficacy and safety, we defined the following Figure 1) is a polyphenol functionalized-D-glucoside-xanth-
primary and secondary endpoints for our investigation. one family of phytochemical found in abundance in the
Anacardiaceae and Gentianaceae family of plant species espe-
Primary Endpoint (Safety) cially in mangoes skin and honeybush tea.56,57
NCI toxicity criteria at every course-adverse events were Mango peel (MP) extracts and mangiferin (MGF), (one
summarized by NCI CTC (Common Toxicity Criteria) of the major phytochemicals in MP), were utilized to
grading and tabulated by severity and relationship to the produce (MP-AuNPs and MGF-AuNPs) gold nanoparti-
study medications. cles. Mango peel cocktails extract-based and MGF

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conjugated gold nanoparticles (AuNPs) were synthesized thus attributing to optimum in vitro and in vivo stability
by redox reactions where electrons from phytochemicals against agglomeration forces.
are used to reduce gold salt to the corresponding gold
Formulation of NSB Drug
nanoparticles (Figures 1 and 2). The production of MP-
NSB drug was formulated by mixing MP-AuNPs with pro-
AuNPs and MGF-AuNPs were confirmed by employing
prietary combinations of plant phytochemicals from Amalaki
extensive characterization tools. It is important to note that
(Emblica officinalis), Amra (Mangifera indica), Haridra
the mangiferin molecule comprises of glucose and xantho-
(Curcumin longa), Babbula (Acacia nilotica), Yashtimadhu
noid (polyphenols) units. Glucose molecule of MGF sup-
(Glycyrrhiza glabra) (Scheme 1). Reproducibility of this
ports to stabilize nanoparticles and hydroxyl groups of the
formulation has been confirmed at small as well as at indus-
xanthonoid unit play a significant role as powerful redu-
trial scales by measuring all the physicochemical parameters
cing agents to reduce the gold salt to the corresponding which are discussed in the following sections.
gold nanoparticles (MGF-AuNPs).
UV-visible spectrophotometric analysis confirmed the sur- Characterization of NSB Drug
face plasmon resonance (SPR) of MP-AuNPs and MGF- The NSB drug was characterized by TEM technique for
AuNPs at ~535 nm (Figure 3), thus confirming the successful qualitative analysis of AuNPs in the drug formulation. The
synthesis of AuNPs. The transmission electron microscopic core size of AuNPs, obtained by TEM, STEM and EELS
(TEM) analysis of MP-AuNPs and MGF-AuNPs indicated indicated that the NPs are spherical, mono-disperse and
that these nanoparticles are spherical, mono-disperse and homogenous with the core size of 35±2 nm (Figure 4).
homogenous with the core size of 35±2 nm (Figure 3). The The solutions of NSB drug were also analyzed for zeta
results obtained by dynamic light scattering instrument potential (−17±5 mV) by dynamic light scattering (DLS)
revealed that MGF-AuNPs showed a hydrodynamic size at instrument.
55±5 nm whereas MP-AuNPs exhibited a size of 65±5 nm. Drug Loading of Phytochemicals from Mango Peel on
Zeta potential obtained for MGF-AuNPs and MP-AuNPs Gold Nanoparticles
indicated large negative values of −40±2mV and −20±2 mV, Mango peel contains a large abundance of mangiferin (42% of
respectively (Table 1). Our observations of hydrodynamic the total polyphenols, Figure 5) – a heat stable and naturally
sizes for both AuNPs which are greater than their core sizes occurring pharmacologically active phytochemical with anti-
(35±2 nm), confirmed the encapsulation of phytochemicals/ inflammatory, antitumor and antioxidant activities.27,58–60 As
MGF onto the gold nanoparticle. The larger negative zeta shown in Figure 5, additional phytochemicals found in mango
potential, observed for MGF-AuNPs and MP-AuNPs, sug- peel in smaller proportions include quercetin, quercetin
gested strong repulsive forces between the nanoparticles, 3-o-rhamnoside, Kaempferol, and Rhamnetin.58

2.5
1 B) MGF-AuNPs
A) MP-AuNPs
2
0.8
Absorbance

1.5
Absorbance

0.6

1
0.4

0.2 0.5

0 0
300 400 500 600 700 800 400 500 600 700 800
Wavelength (nm) Wavelength (nm)

Figure 3 Characterization of AuNPs by UV-Visible spectrum and TEM techniques (A) MP-AuNPs, (B) MGF-AuNPs.

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Table 1 Physicochemical Data Parameters of Gold Nanoparticles (AuNPs)

Sample Absorbance Core Size by Hydrodynamic Zeta Potential [Au] in AuNPs
(nm) TEM (nm) Size by DLS (nm) (mV) (by AAS, ppm)

MGF-AuNPs 535 35±2 55±5 −40±2 327

MP-AuNPs 535 35±2 65±5 −20±2 327
Abbreviations: TEM, transmission electron microscopy; AAS, atomic absorption spectroscopy; DLS, dynamic light scattering; MGF, mangiferin.

As shown in Figures 1, 2 and 5, the antioxidant power of laboratory.19–21,23,26,27,35,61 We hypothesized that the cor-
mangiferin and other phytochemicals in mango peel are ona around gold nanoparticles in MP-AuNPs comprises of
responsible in transforming gold salt into the corresponding mostly the encapsulation of mangiferin due to the large
AuNPs. abundance of this phytochemical in the mango peel
In order to understand the extent of loading of mangi- (Figure 5). Validating this hypothesis is significant in
ferin onto AuNPs, which we refer as drug loading, we understanding drug loading of mangiferin around gold
have performed key experiments as elaborated in numer- nanoparticles. In order to validate this hypothesis, we
ous publications on green nanotechnology from our performed independent reactions of interacting mangiferin

Figure 4 (A) Core size distribution of AuNPs in 'Nano Swarna Bhasma' (NSB) drug by TEM, (B) STEM image shows lattice structure of Au, (C) Graph shows presence of
Au in NSB drug by EELS.

0% 1%2% 2%
2% 3% Mangiferin
5% Mangiferin gallate
Isomangiferin
Isomangiferin gallate
Quercen 3-O-galactoside
42%
Quercen 3-O-glucoside
14%
Mangiferin Quercen 3-O-xyloside
Quercen 3-O-arabinopyranoside
Quercen 3-O-arabinofuranoside
Quercen 3-O-rhamnoside
16% Kaempferol 3-O-glucoside
Rhamnen 3-O galactoside/glucoside
8% Quercen

2% 3%
Figure 5 Pie chart represents polyphenols and various phytochemical compounds in mango peel (mg/Kg) on dry matter basis.

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with gold salt (Figure 2). Complete physicochemical char- MGF per nanoparticle = Number of MGF per mL/number
acteristics of these nanoparticles, in terms of size and of nanoparticle per mL
charge, are summarized in Table 1. We further calculated = 2.5x10 /7.6x10 = 3.3x105
17 11

the number of mangiferin molecules per gold nanoparticle

using the following equations: Determination of Number of MGF per MP-AuNP
The concentration of MGF in MP-AuNPs is 9 μg/mL as
Calculation of Average Number of Gold Atoms per obtained from LC-MS spectrophotometry. Therefore, the
Nanoparticle number of MGF moles per mL is 1.2x1016. Further, the
The average number of gold atoms per nanoparticle is number of MGF molecules per NPs was calculated using
calculated using core size value of the MP-AuNPs and the formula:
MGF-AuNPs obtained from TEM analyses. The average MGF per nanoparticle = Number of MGF per mL/
core diameter of the particles (D, nm) are summarized in number of NPs per mL
Table 1. We found that the core sizes of MP-AuNPs and
MGF-AuNPs were similar. Assuming a spherical shape = 1.2x1016/7.6x1011 = 1.7x104
and a uniform face-centered cubic (fcc) structure, the Stark similarities of physicochemical properties includ-
average number of gold atoms (N) for each nano-sphere ing plasmon resonance, core size, hydrodynamic size – of
was calculated using Eq. 1, where ρ is the density of the MP-AuNPs and MGF-AuNPs (Table 1) – unequivocally
fcc gold (19.3 g/cm3) and M is the atomic weight of gold confirmed that the phytochemical corona constitution of
(197 g/mol) and NA is the Avogadro’s number.62 these two types of nanoparticles are very similar. We have
therefore reasoned that the largely abundant mangiferin phy-
πρD3 tochemical dominates in the formation of phytochemical
N ¼ NA (Eq:1)
6M corona and that the mango peel extracts can be reliably
N = 1.3x106 gold atoms per nanoparticle used to achieve effective mangiferin drug loading around
The calculations as outlined above confirmed that MP- gold nanoparticles. The similarity of physicochemical para-
AuNPs and MGF-AuNPs consisted of 1.3x106 gold atoms meters of MGF-AuNPs and MP-AuNPs also support the
per nanoparticle. hypothesis that the number of mangiferin units per nanopar-
ticle in these two types of nanoparticles are comparable.
Determination of Concentrations of Nanoparticle These drug loading experiments gave us the confidence
Solution to utilize mango peel-derived gold nanoparticles for
The concentration of the nanoparticle solution was calcu- further in vivo therapeutic efficacy studies discussed in
lated based on the amount of NaAuCl4 (100 µL of 0.1 M) the following sections.
used for the respective nanoparticle synthesis. It involves
1x105 moles of gold or 6.0x1018 total gold atoms (NT). Effect of NSB Drug on Cancer and
The concentration of the nanoparticles C was calculated
using Eq. 2 by dividing the total number of gold atoms
Normal Cell Viability
In order to test the in vitro antitumor efficacy of the NSB
(NT) with the average number of gold atoms per nano-
drug, serial dilutions were prepared in DMEM (Dulbecco’s
sphere (N=1.3x106) and V (6 mL) the volume of the
Modified Eagle Medium) media for treatment with tumor
reaction solution.62
cells. The cell viability profile of the NSB drug was
NT evaluated against breast cancer cells (MDA-MB-231) and
C¼ (Eq:2)
NV also with the normal endothelial cells (HAECs) using
= 7.6x1011 nanoparticles/mL MTT assays (Figure 6). The cell viability profiles demon-
strated that the NSB drug exhibited dose dependent effi-
Determination of Number of MGF per MGF-AuNP cacy in cell death of cancer cells. Our results also
The concentration of MGF in MGF-AuNPs is 173 μg/mL confirmed that the NSB drug is selectively toxic to tumor
as obtained from LC-MS spectrophotometry. Therefore, cells with minimal or no toxicity against the normal cells
the number of MGF molecules per mL is 2.5x1017. (Figure 6).
Further, the number of MGF molecules per nanoparticle Based on these encouraging in vitro antitumor efficacy
was calculated using the formula: of the NSB drug, we have further performed in vivo

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120 48 hr the day of randomization and treatment was considered

Cell viability (% of control) as day zero.
100
Results of therapeutic efficacy studies, as summarized in
80
Figure 7, provided conclusive evidence that the NSB drug
60 has the ability to reduce tumor volume. As outlined in
40 Normal endothelial cells Figure 7, after approximately 1 week of treatment (day 10),
20 MDA-MB-231 Cancer cells
the tumor volume appeared to decrease in the treated group
of tumors bearing mice. Four weeks post-treatment (day 24),
0
0 12.5 25 50 100 200 the tumor volume of the treated group was between 0.02
Conc (μg/mL) ±0.01–0.03±0.01 cm3 and continued to decrease signifi-
cantly. Five weeks post-treatment (day 31), tumor volume
Figure 6 Effect of NSB drug on breast cancer (MDA-MB-231) cells viability and
non-toxic nature on normal endothelial (HAECs) cells. of the untreated control group (0.08±0.02 cm3) was signifi-
cantly larger than the tumor volumes of the treated groups
therapeutic efficacy to investigate the ability of this new (0.03 ±0.01–0.04 ±0.01 cm3) (Groups 2–3). NSB drug at the
drug to control or reduce tumor size. We have used mice, doses of 3 and 7 mg/30 gm of animal body weight showed
implanted with breast tumor xenografts of human breast significant reduction in the tumor volumes.
tumor cells (MDA-MB-231), in the therapeutic efficacy Complete analysis of the therapeutic efficacy data has
studies. MDA-MB-231 breast tumor cells are excellent mod- revealed that the tumor volumes for the control group
els for testing the efficacy of new therapeutic agents for remained larger than the treated groups throughout the
treating triple-negative breast cancers in human patients.63 course of therapeutic efficacy investigations. Therefore,
our results demonstrate that the NSB drug has significant
therapeutic efficacy as reflected in its ability to control/
In vivo Therapeutic Efficacy reduce the tumor volumes by over 80% (during 60 days of
The in vivo therapeutic efficacy study was conducted in
treatment; Figure 7) as compared to the control groups.
three groups of mice bearing breast tumors with compar-
able size. The mean tumor volume of these groups ranged
from 0.01 to 0.02 cm3 and group mean body weights In vivo Toxicity Studies of Nano Swarna
ranged from 24 to 30 g. The animals (group 2 and 3) Bhasma (NSB) Drug
were given NSB drug twice per week (in peanut butter) The body weight of all the experimental animals was
whereas the first group was given peanut butter without measured to evaluate the toxicity of the NSB drug. The
the drug which served as an untreated control group. The drug was given orally twice per week for 60 days. The
in vivo study was performed over a period of 8–9 weeks; animals were monitored for their body weight and health

0.90
0.80
0.70 Untreated control
Tumor Size (cm3)

0.60 NSB-3
0.50 NSB-7
0.40
0.30
0.20
0.10
0.00

Days after palpable tumors randomized

Figure 7 Therapeutic effects of NSB drug to control or reduce tumor size in human breast tumor bearing SCID female mice. Animals randomized and treated orally on day
zero. Treatment was given twice per week; n=7.
Note: Untreated control group represents no treatment, only tumor bearing SCID mice.

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conditions during the treatment period. Body weights were B (standard of care treatment along with the NSB drug)
measured twice per week for all the groups. Animals were was reduced at week 7 and further reduced at week 12, in
sacrificed at the end of the study or when tumors reached comparison with the baseline measurements. Whereas in
endpoint. The body weights of NSB drug treated animals the other treatment group (arm A which received Standard
were found to be similar to that of the normal control of Care drugs), where the patients were treated only with
group (Figure 8). Overall, the detailed analysis, as outlined the “standard of care treatment”, drastic increase in the
in Figures 7 and 8, demonstrates that NSB drug did not mean sum of the longest diameter was observed at week 7,
cause any adverse systemic toxicity throughout the course which reduced at the last visit with respect to the baseline
of the treatment regimen. measurements. Mean SD of treatment arm B (3.31) was
There are no effective FDA approved drugs so far that much higher as compared to that of treatment arm
control the growth and proliferation of triple-negative breast A (1.37), i.e. lesion sizes of treatment arm A were of
tumors. The results of the therapeutic efficacy studies using the high variability. Mean SD of treatment arm A increased
NSB drug, as shown in Figure 7, are highly significant because to 4.41 (percent change from baseline 65.00) at visit 4 and
doses in the range of 3–7 mg per 30 gm mouse, administered 3.78 (percent change from baseline 59.64) at visit 6. On
two times a week, are able to control the growth and prolifera- the other hand, higher mean SD of treatment arm
tion in a highly effective way as compared to the untreated B reduced to 3.00 (percent change from baseline 18.22)
control groups. The breast tumor model used in our investiga- at visit 4 and slightly increased to 3.64 (percent increase
tion is a highly aggressive tumor classified to be triple-negative from baseline 28.38) at visit 6. These observations suggest
. Our results, therefore, provide conclusive experimental evi- that the percent change from baseline in SD of the treat-
dence that the NSB drug can be used in humans for treating ment group arm A was more than double that of the
breast tumors to relieve tumor burden and to enhance the life treatment arm B at the end of the treatment.
expectancy and quality of life of patients. Therefore, regulatory Tumor response was calculated based on RECIST 1.1
permission to conduct pilot clinical trials in human breast for all completed patients. There were no non-target
cancer patients was sought from the Indian Ayurvedic drug lesions diagnosed in any of the patients. Therefore,
regulatory agency AYUSH. Results of the pilot clinical trials response in target lesions corresponds to overall response.
are described below. None of the patients exhibited complete response during
the study. Two patients (66.7%) in treatment arm A and
one (33.3%) in treatment arm B showed partial response at
Results of Pilot Clinical Trial week 12 of evaluation. Two (66.7%) patients showed no
Investigations change in the lesion. i.e. stable disease in treatment arm
Efficacy B at week 12 evaluation. Progressive disease response was
Efficacy was a secondary objective of the study. Mean sum observed in one patient (33.3%) throughout the treatment
of the longest diameter of target lesion in treatment arm period of treatment arm A, whereas none of the patients
showed progressive disease in treatment arm B.
31 At the end of the treatments, i.e. week 12, clinical benefit
Untreated control NSB-3 NSB-7 Control
29 rate/disease control rate was calculated for all the patients.
Patients treated with NSB drug along with the “standard of
Body weight (Grams)

27
care treatment” (arm B) exhibited 100% clinical benefit rate
25 when compared to the treatment group arm A which received
23 only the “Standard of Care Treatment”.

21
Safety
19 Out of the total enrolled patients, two patients each from
0 3 7 10 14 17 21 24 28 31 35 38 42 45 49 52 56 60 63
Days both the A and B groups experienced at least one adverse
event. Majority of adverse events (07 AEs) were mild in
Figure 8 Effect of NSB drug on body weight of SCID mice after oral administration
in human breast cancer bearing SCID mice. severity. Only one AE reported by the patient in treatment
Note: Untreated control group represents no treatment, only tumor bearing SCID
mice, and control group represents no treatment and no tumor bearing mice
arm B was severe in nature. More than 50% of total
(healthy control mice). reported AEs were from treatment arm A, out of which

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05 were possibly related and one each was unlikely related with herbal formulation NSB drug in breast cancer. Seven
and unrelated to the study drug. There were 04 incidences female patients with pathologically confirmed invasive breast
of Neutropenia of mild severity reported by one patient carcinoma stage IIIA or IIIB were enrolled in the study. One
from treatment arm A followed by hyperacidity and weak- patient was withdrawn at baseline visit due to non-
ness generalized reported by two patients, one each from compliance. Out of the remaining six patients, three patients
both the treatment arms. Anemia was reported for one were treated with treatment A and three were treated with
patient from treatment arm B, severe in nature but not treatment B. All six patients completed the study and received
related to the study drug. full dose of treatment in both groups A and B as per rando-
mization. Safety was the primary objective of the study.
Safety analysis was done on safety population. There were
Discussion Related to Pilot Clinical no alarming toxicity effects observed in both the groups.
Trial Investigations Tumor response was calculated for patients who com-
Challenges continue in the treatment of the three most common pleted clinical trials. There were no non-target lesions diag-
types of breast cancer subtypes: estrogen-receptor-positive nosed in any of the patients. Therefore, response in target
(ER+), human epidermal growth factor receptor 2-positive lesions corresponds to overall response. None of the patients
(HER2+), and the more heterogenous triple-negative breast exhibited complete response during the study. Two patients
cancer (TNBC) diseases.64,65 Treatments focused on targeting (66.7%) in treatment arm A and one (33.3%) in treatment
the molecular mechanisms of endocrine resistance in ER+ arm B showed partial response. Two (66.7%) patients
advanced breast cancer are evolving with some success.66–69 showed no change in the lesion. i.e. stable disease in treat-
Although several targeted treatments including Afinitor, ment arm B. Progressive disease response was observed in
Avastin, Herceptin, Ibrance, Kadcyla, Kisqali, Lynparza, one patient (33.3%) of treatment arm A. None of the patients
Nerlynx, Perjeta, Tykerb, and Verzenio – are in clinical practice in treatment arm B showed progressive disease.
for treating patients with HER2+ breast cancer, the disease Figure 9 is a waterfall plot that presents each individual
continues to show considerable mortality within the patient patient’s response to the drug based on parameters, such as
population around the world.70–72 In particular, the targeted lesion size (sum of longest diameter) and its change from
agents have had much less effect in patients with triple- the baseline. One patient in treatment arm A is a non-
negative breast cancer. Poly ADP ribose polymerase (PARP) responder whereas all three patients in treatment arm B are
inhibitors are currently the most promising agents in this group, responders. This means that all the patients treated with
based on the similarity of this subtype to ovarian cancers.73–77 treatment arm B showed 100% disease control rate (DCR)
Alternative holistic approaches are being continuously sought or clinical benefit rate (CBR). Patients treated with treat-
as the World Health Organization (WHO) forecasts that ment arm A showed 66.7% CBR (Table 2).
approximately 80% of the global population would use one Efficacy analysis was done on FAS population. At the end
or the other form of alternative medicine in their life time.78 of 12 weeks, none of the patients showed complete response.
In this report, we have described the application of green No patient treated with NSB drug (DNANOSTANNA) cap-
nanotechnology to holistic Ayurvedic medicine. Our discov- sules showed progressive disease. Patients from this group
ery of Nano Swarna Bhasma (DNANOSTANNA capsules) exhibited 100% clinical benefit rate.
through green nanotechnology as outlined in Figure 1, 2 and
Scheme 1 is one such formulation containing gold nanopar- Conclusions
ticles encapsulated, with a cocktail of phytochemicals of Overall, we have been able to clinically translate the findings
gooseberry, mango, turmeric and gum arabic – all approved from the pre-clinical investigations of the Nano-Ayurvedic
for use in the Ayurvedic medicine modality. The compelling medicine gold nanoparticles-based drug to demonstrate
in vitro antitumor results and the in vivo therapeutic efficacy acceptable safety and excellent efficacy results in human breast
of the NSB drug prompted us to obtain regulatory approval cancer patients. Administering the Nano Swarna Bhasma (
from the Indian Ayurvedic medicine AYUSH regulatory NSB) drug (DNANOSTANNA in the form of capsules) to
authority to initiate pilot clinical trials in human breast patients with breast cancer did not show adverse effects as
cancer patients. monitored through both vital signs and laboratory chemistries.
In this open label, controlled study, “Standard of Care Commonly reported adverse events were transient implying
Treatment” was compared with the same treatment combined acceptable safety. Toxicity/adverse event assessment by NCI

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Figure 9 Waterfall plot for percent change from baseline in tumor’s sum of longest diameter at week 12 by treatment.
Notes: Patients in treatment arm A received the following- Doxorubicin (60 mg/m2 every 3 weeks as a 1 hr intravenous infusion) and Cyclophosphamide (600 mg/m2 every
3 weeks as a 1 hr intravenous infusion). Patients in treatment arm B received the following- Doxorubicin (60 mg/m2 every 3 weeks as a 1 hr intravenous infusion),
Cyclophosphamide (600 mg/m2 every 3 weeks as a 1 hr intravenous infusion) and NSB Drug– two capsules thrice a day after food.

toxicity criteria performed at every visit certainly provided Bhasma can be safely used as a valuable adjuvant therapeutic
clinical evidence on the potential of this therapy containing agent to reduce the adverse effects of routine chemotherapeutic
Nano Swarna Bhasma-gold nanoparticles encapsulated with agents while providing measurable therapeutic efficacy in
a proprietary combination of various Ayurvedic herbs treating breast and other forms of human cancers. Green nano-
(Scheme 1). Our results also indicate that Nano Swarna technology-based Nano-Ayurvedic formulations, such as NSB
drug, show minimal toxicity to normal cells and tissues.
Table 2 Summary of Overall, Target and Non-Target Lesion Therefore, Nano-Ayurvedic medicine modalities present rea-
Response at Week 12 by Treatment listic opportunities as holistic and highly effective interven-
Tumor response Arm Arm Total tions in the care and treatment of cancer patients globally.
A (N=3) B (N=3) (N=6)
n (%) n (%) n (%)

Overall response Ethics Approval and Informed

Complete response 0 (0.0) 0 (0.0) 0 (0.0)
Consent
Partial Response 2 (66.7) 1 (33.3) 3 (50.0) This study, including the procedures for patient enrollment
Stable Disease 0 (0.0) 2 (66.7) 2 (33.3) and recruitment, was approved by the Institutional Review
Progressive Disease 1 (33.3) 0 (0.0) 1 (16.7) Board of two independent hospitals (Ashwin Hospital,
Target lesions Chennai, India and Erode Cancer Institute, Erode, India).
All patients, who participated in the study provided written
Complete response 0 (0.0) 0 (0.0) 0 (0.0)
informed consent. This study was conducted in accordance
Partial Response 2 (66.7) 1 (33.3) 3 (50.0)
Stable Disease 0 (0.0) 2 (66.7) 2 (33.3)
with the approved guidelines of the government of India
Progressive Disease 1 (33.3) 0 (0.0) 1 (16.7) regulatory authority of Ayurveda, Yoga and Naturopathy,
Unani, Siddha and Homoeopathy (abbreviated as AYUSH).
Non-target lesions
This study was conducted in accordance with the ethical
Complete response 0 (0.0) 0 (0.0) 0 (0.0) principles laid down by Declaration of Helsinki (Taipei
Stable Disease 0 (0.0) 0 (0.0) 0 (0.0)
2016) following the principles of ICH Guidelines for Good
Progressive Disease 0 (0.0) 0 (0.0) 0 (0.0)
Clinical Practice (1997) and Schedule.

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Acknowledgments 14. Risberg T, Kolstad A, Bremnes Y, et al. Knowledge of and attitudes

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acknowledged. We also acknowledge the support of the 17. Tandon N, Yadav SS. Contributions of Indian Council of Medical
contract research organization, 15 Clinical Research, Pvt. Research (ICMR) in the area of Medicinal plants/Traditional medicine.
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