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Hirak Bhasma: A Potential Ayurvedic Antibacterial Drug Assessed by In Vitro Pre-Clinical Studies

SUTAPA SOM CHAUDHURY,1 BHUBAN RUIDAS,1 PRASANTA KUMAR SARKAR,2 and CHITRANGADA DAS MUKHOPADHYAY1

Center for Healthcare Science and Technology,

Indian Institute of Engineering Science and Technology, Shibpur, Howrah - 711103, West Bengal, India,

E-mails: This email address is being protected from spam bots, you need Javascript enabled to view it (S. S. Chaudhury), This email address is being protected from spam bots, you need Javascript enabled to view it (C. D. Mukhopadhyay)

2Department of Rasashastra, ]. B. Roy State Ayurvedic Medical College and Hospital, Kolkata - 700004, West Bengal, India

ABSTRACT

The growing evidences of multi drug resistance are of genuine concern to combat nosocomial diseases in current time. Herein this study Hirak Bhasma (HB), the widely used herbometallic drug in Ayurveda, was prepared as a nano-drug component and evaluated thoroughly in vitro for the pre-clinical evaluation of an effective anti-bacterial drug candidate. HB showed rich mineral constituents along with some below detection level toxic elements such as Si, Ni and A1 which enhance its potential as an antibacterial drug. The nano-dust preparation of the HB came with the presence of a higher organic compositions and mineral constituents having a particle size in nanometer range as was evidenced by the physic-chemical characterization via inductively coupled plasma optical expression spectrometry (ICP-OES), Fourier transform infrared spectra (FTIR), and field emission scamring electron microscopy (FE-SEM) techniques respectively. Antimicrobial potential of

HB was tested in clinically isolated pathogenic strains of Escherichia coli, Staphylococcus aureus and Candida intermedia through disc diffusion assay, broth turbidity measurements and cell imaging via FE-SEM. The non-cyto- toxic dose of HB was found to be 50 pg/ml by the reduction of tetrazolium dye MTT (3-(4,5-dhnethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT assay) on the human breast cancer cell line MCF7. HB was effective in increasing the intracellular reactive oxygen species (ROS) in the bacterial cells while being helpful to keep the intracellular redox balance in human cells. This proves that HB has the potential to be the effective anti-bacterial drug for the treatment of diseases caused by bacteremia with a minimal or no side effect. Moreover, in the context of the evaluation of the ROS level, HB appeared as a promising drug candidate for the anti-inflammatory and immune-modulatory effects although it has not shown any antifungal effect.

Since long the herbometallic ayurvedic compound HB claims its high potential as adaptogenic antibacterial agent, analgesic, antimicrobial, alternative antioxidant, anti-inflammatory, immunomodulator and so on; but there is lack of scientific evidences. This work reports the lagging scientific documentation for HB as antibacterial and anti-inflammatory agent.

INTRODUCTION

In the current scenario, the multi-drug resistance (MDR) of pathogenic bacterial strains has already rung an alarm to the treatment of nosocomial infections [1]. The overuses of broad-spectrum antibiotics and their prolonged applications are making the situation worsen day by day [2, 3]. Since last, few decades’ combinatorial therapies are being proposed to combat MDR bacterial strains [4]. But the use of antibiotics in combinations is also in vain because the question has already been raised whether we are currently in the post-antibiotic era [5]. The increasing resistance to the antibiotics available until date necessitates the use of biocompatible natural compounds as an alternative medicine to combat bacterial pathogenesis without any side effects. Nowadays natural compound based Ayurvedic preparations are widely accepted as an effective antimicrobial drug [6, 7]. In Ayurveda, the traditional Indian medical system, Hirak Bhasma (HB) is such a preparation. This Ayurvedic medicine is mainly composed of Hirak,

i.e., diamond dust. HB is prescribed for treating immunity disorders, crippling rheumatoid arthritis, bone marrow depression, cancer, and so on [8]. Here, we report a novel preparation of HB as a nano-drug component and its thorough in vitro evaluation for the pre-clinical assessment as an effective anti-bacterial drug candidate. Although, it has been claimed that HB is highly potential as an adaptogenic, antibacterial, anti-inflammatory, and immunomodulatory drug, no scientific evidence is available in support of this claim [9]. Metallic sources including sulfide-bearing minerals, metal oxides, and alumina silicates like biotite mica, chalcopyrite, and others have been used in traditional preparation as metallic antimicrobial sources. Interestingly, the antimicrobial function of metallic preparations is best achieved when the metal ions are effectively blended with the organic molecules [10,11]. None the organic compounds alone or the metals singly can construct a highly effective antimicrobial drug. Ag, Au nanoparticles have also been in the limelight since few decades but anyone metal formula as a sole composition of an antimicrobial drug candidate may appear with the drawbacks of intense side effects [12, 13]. Destruction of pathogenic microbes via an elevated level of reactive oxygen species (ROS) is one of the prime mechanisms of these metal-based drug candidates, but this comes with the uncontrolled damage of the neighboring healthy cells in patients [14]. Here lies the importance of a perfect blend of organic compounds along with the trace metal components as in the HB. Being a balanced source of mineral components such as Zn, Cr which is effective against pathogenic microbes, the nanoformulation of HB may pave the path towards an alternative medicine in this premise [15, 16]. Also, HB is prescribed in Ayurveda to rejuvenate the body and mind [17]. This seems to be an advantage to regain the potentiation of the body in the disease condition such as bacteremia. But these claims need a scientific support. This work reports the lagging scientific documentation for HB as an antibacterial agent.

MATERIALS AND METHODS

10.20.1 PREPARATION OF HIRAK BHASMA (HB)

The HB (incinerated diamond powder) was procured from J. B. Roy State Ayurvedic Medical College and Hospital, Kolkata, West Bengal, India. The raw diamond powder was collected from Diamond Market, Surat, India. Briefly, the purification treatment of raw diamond powder was done by heating the powder to red hot and then quenching immediately in horse gram (Dolichosbiflorus) decoction. The procedure was repeated for 21 times. The obtained powder was taken in a mortar; levigation was done by horse gram (Dolichosbiflorus) decoction for six hours; pellets were prepared and dried. Those were taken in between two earthen saucers and the junction was sealed. The arrangement is called Sharava Samputa in parlance of Ayurveda. It was placed inside a muffle furnace and heating was done at 750°C for 1 h. After self-cooling, the pellets were collected and made into powder form. Levigation by horse gram decoction and heat treatment were done for 25 times. The powdered drag was further purified by sequential soxlilet extraction process with hexane, dichloromethane, and methanol and stored in a glass bottle (Scheme 10.1 and Figure 10.1). The final yield was about 10-15% of the initial raw material.

The image of procured HB powder

FIGURE 10.1 The image of procured HB powder.

SCHEME 10.1 Flow chart representing preparation of Hirak Bhasma.

The solvents for the soxhlet extraction and all the other reagents were of analytical grade and commercially procured from Sigma-Aldrich (MO, USA) if not mentioned in the text.

10.2.2 PHYSICO-CHEMICAL CHARACTERIZATION OF HB

The total cation concentration of HB was screened with the aid of inductively coupled plasma-optical emission spectra (inductively coupled plasma optical emission spectra (ICP-OES); Thermo iCAP 7000 series). By analyzing the Fourier transform infrared spectrum (FT-IR) (Perkin-Elmer Spectrum 100 FT-IR spectrometer) the presence of organic fractions were detected in HB compound. The average particle size and morphology of HB was recorded under thefield emission scanning electron microscopy (FE-SEM) (Carl Zeiss Supra SEM instrument). The pH of the HB solution was recorded with the aid of a pH meter (Eutech pH 700).

10.2.3 DRUG RESISTANCE PROFILE OF THE CLINICALLY ISOLATED BACTERIA

In this study, two clinically isolated bacterial strains of Escherichia coli and Staphylococcus aureus, and one fungal strain of Candida intermedia were chosen to prove the antimicrobial action of HB. The MDR characteristic of these microbes was also confirmed following a standard protocol reported elsewhere [7]. Briefly, stock solutions of 100 pg/ml oxacillin, ampicillin, chloramphenicol, gentamycin, tetracycline, and levofloxacin each were prepared and diluted to a final concentration of 40 pg/ml. Approximately 20 ml LB agar (HiMedia Laboratories, India) containing these antibiotics each at their final concentration were plated. Then, a 100 pi of 10s CFU/rnl log phase culture of the respective bacteria were spread on those agar plates and grown at 37°C for 24 h. Bacterial growth was measured in tenns of the percentage of the colony-forming unit with respect to the initial inoculum. A plate containing 40 pg/ml of HB was compared with respect to those containing antibiotics.

10.2.4 ANTIMICROBIAL ACTIVITY ANALYSIS

The in vitro antibacterial ability of the HB was appraised using the disc diffusion method following the protocol by Zaidan et al. [20]. HB extract was tested (at 25 pg/ml-10 mg/ml) against clinically isolated E. coli, S. aureus, and C. intermedia by incubating them at 37°C for 24 li in LB agar and yeast extracts peptone dextrose (YPD) agar (HiMedia Laboratories, India), respectively keeping ampicillin as the positive control. Broth turbidimetric analyses were performed following a modified method by Berridge and colleagues to determine minimal inhibitory concentration (MIC) via the measurement of OD600 [21]. From the tubes where the respective cultures were first inhibited (determined by the absence of visible growth) by HB a 100 pi broth was taken and spread on a fresh LB agar plate each. Thus, the minimal bactericidal concentration (MBC) was determined. In addition, a bacterial cell viability assay was performed with an unproved MTT assay, i.e., 3-(4,5-dimethylthi- azol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay (MTT assay kit, Invitrogen, Thermo Fisher Scientific Corporation) [22].

10.2.5 IMAGE DOCUMENTATION OF HB TREATED MICROBES

FE-SEM was performed to visualize morphological changes of drug treated microbes at different time interval compared to untreated microbes as control. Drug treated bacterial smear was heat-fixed and dipped in formaldehyde for 2 h at RT. After washing and proper dehydration, samples were dried in vacuum for 2-3 hours at RT and imaged by FE-SEM.

10.2.6 CYTOTOXICITY ASSAY

The non-cytotoxic dose of HB was checked by MTT assay on the human breast cancer cells MCF-7 reported elsewhere [23]. Briefly, 10,000 cells maintained in high glucose Dulbecco’s modified eagle media (DMEM) (HiMedia Laboratories, India) along with 10% FBS and IX penicillin-streptomycin were seeded per well of 96 well plates. The HB was added and tested over the concentration range of 10 pg/ml to 100 pg/ml. After 24 h treatment, the media was changed and 10 pi MTT solutions (1 mg/ml) were added to each well. It was incubated for 4 h and the resulting Formosan crystals were solubilized in dimethyl sulfoxide (DMSO) prior to the absorbance measurement at 570 nm wavelength.

10.2.7 INTRACELLULAR ROS MEASUREMENT

The increase in intra-cellular ROS with an increasing concentration of HB was measured by the fluorescence DCFDA method. For this, MCF-7

cells were treated with HB with an increasing concentration of 10-100 pg/ml for 12 h at 37°C and 5% CO,. Following a washing step with IX ice-cold PBS the cells were again incubated with 100 pM DCFDA (Invitrogen, Thermo Fisher Scientific Corporation) for 30 min in the dark at 37°C. The fluorescence intensity was measured spectroscopically with the excitation and emission wavelengths at 485 nm and 520 nm, respectively [24].

10.2.8 CHANGES IN CELLULAR ROS IN BACTERIAL CELLS

Change in the intracellular ROS after drug treatment in microbes was evaluated following the protocol by Su et al. via the DCFHDA method, to determine the mechanism of the drug action [25]. Briefly, 4 ml LB broth was inoculated with 2% log-phase bacterial culture and treated with 10-100 pg/ ml HB at 37°C for 1 h. To this bacterial culture 10 pM DCFHDA (Invitrogen, Thermo Fisher Scientific Coiporation) was added and incubated in dark for 30 min before recording the emission spectra at 529 mn with the excitation wavelength at 504 nm.

RESULT AND DISCUSSION

The presence of major inorganic elements like Fe, Al, and Ca was documented by the ICP-OES study (Table 10.1). The bacterial growth inhibiting influential elements were Ni, Si, Cr, and Zn, which were present as parts per million (ppm) levels in the final nano preparation of HB. These implied low toxicity of HB as a drug candidate as expected (Figure 10.7). Moreover, these heavy metals altogether may impact a synergistic effect to bring out the antibacterial action against pathogenic E. coli and S. aureus tested.

The Bhasma are claimed to be biologically produced nanoparticles [19]. The organic compounds present in HB were detected via the absoiption peaks (between 650-1020 cm-1 due to C-H and C-N group, between 1410-1420 cm-1 due to C-O, C-CH, and N-H group and between 3200-3600 cnr1 due to OH group) obtained horn FTIR study (Figure 10.2A). This proved the presence of organic components along with the cationic metals (i.e.,Fe, Ca, Cr, Zn, Al, etc., detected in ICP-OES) in HB preparation. The average diameter of the HB nanoparticles was about 30 nm as depicted by the FE-SEM analyses (Figure 10.2B).

TABLE 10.1 ICP-OES Analysis has Shown the Total Cation Concentration of the HB

Element

Concentration (ppm)

Fe

730.48

A1

270.592

Ca

70.355

Ni

16.05

Si

8.03

Cr

28.007

Zn

32.79

FTIR profile of HB (A) and FE-SEM micrograph showing the particle size of HB (B)

FIGURE 10.2 FTIR profile of HB (A) and FE-SEM micrograph showing the particle size of HB (B).

HB has shown an antibacterial activity at a lower concentration of 50 pg/ml in the disc diffusion method (Figure 10.ЗА and B). HB did not show any anti-fungal activity on testing with C. intermedia (data not shown).

By testing the sensitivity of the bacterial cultures for commercially available antibiotics, it was observed that the clinically isolated strains of E. coli and S. aureus were moderately resistant to the penicillin group of antibiotics and also to the protein synthesis inhibiting antibiotics like chloramphenicol, tetracycline, and gentamycin. Although in comparison with the above- mentioned antibiotics, HB has shown a greater efficiency as a bactericidal drug candidate (Table 10.2).

Zones of inhibition against£. coli (A) and S. aureus (B) forthe concentrations of 50-200 |ig/ml of HB with respect to the positive control ampicillin (200 pg/ml)

FIGURE 10.3 Zones of inhibition against£. coli (A) and S. aureus (B) forthe concentrations of 50-200 |ig/ml of HB with respect to the positive control ampicillin (200 pg/ml).

TABLE 10.2 The Antibiotic Resistance Profile of the Clinically Isolated Bacterial Strains Used in the Study

Antibiotics Tested

% Viability of Bacterial Strains (CFU/inl)

E. coli

S. aureus

Oxacillin

45.9

26.08

Ampicillin

26.5

20.8

Chloramphenicol

43

65.7

Gentamicin

39

45.2

Tetracycline

23.8

29.3

Levofloxacin

69.7

87

HB

12.6

17.5

The MIC of HB was 46 pg/ml against E. coli and 115 pg/ml against

S. aureus, respectively when tested over a concentration range from 10-500 pg/ml in broth dilution method of MIC (Table 10.3). The MBC of HB for E. coli and S. aureus was determined as 50 pg/ml and 130 pg/ml, respectively (data not shown here). Interestingly, HB was equally effective against both the Gram-positive S. aureus and Gram-negative E. coli. This signified the good penetration ability of HB irrespective of the thick peptidoglycan layer of Gram-positive bacterial cells. The nano-sized particles of HB with an average 30 nm diameter were advantageous to penetrate the bacterial cells.

TABLE 10.3 The MIC of HB was Found as 46 pg/ml and 115 pg/ml Against E. coli and S. aureus, respectively as Measured by the OD^ Value in the Broth Dilution Method*

Concentration of HB (pg/ml)

Negative Control

OD600£. coli

OD600 .S', aureus

10

0.004

0.59

0.48

20

0.09

0.47

0.39

30

0.008

0.33

0.27

40

0.04

0.18

0.22

50

0.06

0.17

100

0.009

0.12

200

0.01

300

0.01

400

0.004

500

0.006

* LB broth was kept as negative control.

The improved MTT assays for the above-mentioned bacteria (Figure 10.4) and SEM images of the drug-treated microbes also supported this antibacterial activity of HB (Figure 10.5A-D).

The probable mechanism for the antibacterial activity of HB might be the increment in intracellular ROS level in the bacteria (Figure 10.6A). Also, HB was able to increase the intracellular ROS level in MCF-7 cancer cells (Figure 10.6B). This suggested the triggering of the downstream cell signaling cascades leading to the destruction of pathogenic gram-positive and gram-negative bacterial cell walls as well as the controlled demolition of cancer cells. Thus, we can say that HB may influence the potentiation of human body by regulating the immunomodulation and destroying the pathogens present in the body in disease condition.

HB was proved to be a safe drug candidate as its IC50 value was quite high (50 pg/ml) when tested on MCF-7 cell line (Figure 10.7).

CONCLUSION

In conclusion, the presence of various mineralogical constituents of HB has probably enhances the effective action against the bacterial strains synergistically. Most of the element individually plays an important role with effective interference in living system. Thus, the presence of

Time course profile of MTT reduction time for E. coli and S', aureus

FIGURE 10.4 Time course profile of MTT reduction time for E. coli and S', aureus.

FE-SEM image of control S. aureus (A), HB-treated S. aureus at 24 hours (B), control E. coli (C) and HB-treated E. coli at 24 hours, (D) Scale bar is of 2 pm

FIGURE 10.5 FE-SEM image of control S. aureus (A), HB-treated S. aureus at 24 hours (B), control E. coli (C) and HB-treated E. coli at 24 hours, (D) Scale bar is of 2 pm.

Increase in intracellular ROS level in E. coli and S. aureus (A) and in MCF-7 cells (B), with the increase in HB concentration

FIGURE 10.6 Increase in intracellular ROS level in E. coli and S. aureus (A) and in MCF-7 cells (B), with the increase in HB concentration.

different elements together might have played significant role to enhance ROS generation which in turns induces the oxidative stress leading to cell death. Moreover, Enhancement in ROS level may influence the internal signaling cascade directly which is actively associated with cell wall synthesis in both Gram-positive and Gram-negative bacteria. Therefore, a significant ROS enhancement in bacterial cells is a key mechanism of HB mediated cell wall destruction irrespective of the Gram character of the bacteria.

The cytotoxicity of HB. The IC value of 50 |ig/ml has shown the non-toxic nature of HB

FIGURE 10.7 The cytotoxicity of HB. The IC50 value of 50 |ig/ml has shown the non-toxic nature of HB.

FUTURE PROSPECT

The age-old claims of HB to be effective against many diseases including cancer, bacteremia, inflammatory diseases, and neurological disorders and to rejuvenate the body and mind needs more precise scientific documentations. Our report here may file the evidences for the antibacterial profiling of HB leaving a hint on the anticancer and immunomodulatory role of HB. We suggest a future endeavor of the immunomodulatory role of HB in neurodegenerative diseases like Alzheimer’s and Parkinson’s disease (PD) which still lack a therapeutic drug and are coping up with the symptomatic drugs marketed so far.

KEYWORDS

  • field emission scanning electron microscopy
  • Fourier transform infrared
  • Hirak Bhasma
  • inductively coupled plasma optical emission spectra
  • minimal bactericidal concentration
  • multidrug resistance

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