Anti-vibrio effects of the precious Tibetan pill, Rinchen

Tibetan precious pills are an integral part of TTM (Traditional Tibetan Medicine). Among them, Rinchen Drangjor Rilnag Chenmo (RDRC) has been named “King of Precious Pills” due to its efficacy in treating a multitude of human disorders. RDRC has a complex formulation with about 140 ingredients, mostly from medicinal plants and a few precious stones and metals. Not many studies have been done on the experimental validation of antimicrobial properties of this important pill. The current study investigated the antimicrobial activity of the extracts of RDRC. Both aqueous and chloroform extracts were evaluated for their antibacterial potential against a total of seven different bacterial species, which are pathogenic, including three species of Vibrio, viz. V. vulnificus, V. parahaemolyticus and V. harveyi using the well-diffusion method and also by assessing MIC and MBC values. Its antifungal potential was also studied against two fungal strains Aspergillus niger and Talaromyces islandicus. It was found that the chloroform extract of RDRC exerted a positive antibacterial effect on all the Vibrio species tested, and the least MIC of 3.33 mg/ml was observed for V. parahaemolyticus. This is the first study of its kind on the anti-Vibrio effect of the Tibetan precious pill, Rinchen Drangjor Rilnag Chenmo.


Introduction
Traditional Tibetan Medicine (TTM), also known as Sowa-Rigpa in Tibetan (literally translates to "Science of Healing'') is one of the oldest traditional medical systems dating back to the 4th century (1) and world's first integrative medicine (2) with its influence from Chinese, Greco-Arab, Himalayan and Indian medical systems in the 8th century (3). Since then, this holistic and comprehensive medical system has developed as the main medical system in Tibet. Currently, TTM has gained its popularity in India, Nepal, Bhutan, Mongolia, China and western countries. In September 2010, Indian Government officially recognized the Sowa-Rigpa as an integral part of its national healthcare system, making it the fourth country after China, Bhutan and Mongolia to recognize it (4). One of the most important precious pills in Traditional Tibetan Medicine is Rinchen Drangjor Rilnag Chenmo (RDRC). It is considered the "King of Precious Pills'' among the eight precious pills (6). It has been proposed to cure a multitude of diseases and also has rejuvenating effects on the healthy. It is believed to enhance complexion and clear sense organs. It is also a rejuvenator, acts as an aphrodisiac, strengthens nerves, blood vessels and bones (6). RDRC is considered to have prophylactic effects too (6). It is given for a range of diseases from simple to complex and diseases caused by environmental pollution (5). It is also used as a tonic and worn as a protective amulet. RDRC is found to have about 140 ingredients, including detoxified Mercury or Tsothel. It also contains powder from precious stones and metals such as gold, silver, copper, iron, sapphire, diamond, emerald, turquoise etc (6). Some of the important medicinal plants used to prepare these pills are Crocus sativus Linn., Myristica fragrans Houtt., Phytolacca esculenta van Houtte., Terminalia bellerica Roxb. and Terminalia chebula Retz. (6). The preparation method of RDRC has been done sacredly by only a few Tibetan masters and is created on the basis of the practical instructions of the great ancient Masters of Tibetan medicine and enriched with their spiritual blessings. RDRC has been predicted to cure diseases of the gastrointestinal tract, infectious fevers, colic in the small intestine and even cancer (6). It has also been found to eliminate grey hair, wrinkles and is also known to strengthen bones. In China, RDRC has made it to the UNESCO inspired 'Intangible cultural heritage list' in 2006 (7,8). Since its recognition in India from 2010, no work has been done to scientifically validate its claim that is to treat infectious diseases, there is not one single scientific paper on this pill to back up its claim. Therefore, we decided to test the efficacy of both the chloroform and aqueous extracts of RDRC

Sample collection
Rinchen Drangjor Rilnag Chenmo (RDRC) pills were bought from the Men-Tsee-Khang branch clinic in Koramangala, Bangalore, after permission from the Director of Men-Tsee-Khang (Tibetan Medical and Astro-science Institute), Himachal Pradesh, India.

Microbial strains
Staphylococcus aureus (ATCC 29213) and Bacillus velezensis (MW219533.1) were used as the bacterial indicator strains for Gram-positive bacteria. Escherichia coli (ATCC MG1655), Pseudomonas aeruginosa (ATCC 27853), Vibrio harveyi, Vibrio parahaemolyticus and Vibrio vulnificus were used as the bacterial indicator strains for Gram-negative bacteria for antibacterial studies. Aspergillus niger (MT123512. 1) and Talaromyces islandicus (MT123786.1) were used for checking antifungal activity. Some cultures have been maintained in the University and few were a gift from IISc, Department of Cell and Molecular Biology. The culture collection details are given in brackets.

Extraction process
10 gm of pulverized pills were packed uniformly inside a thimble, and a sample was extracted using 120 ml chloroform in a distillation flask at the boiling point of chloroform (62 °C). During the process, condensated fresh chloroform gradually fills the thimble holder, and once it reaches the overflow level, the solute is aspirated by siphoning from the thimble holder back to the distillation flask with extracted analytes (9). This process was repeated for 7 hrs until the extractant became colourless. Because of the large amount of solvent used, the concentration step was essential after extraction using Rotary evaporator (Royal Scientific 137). The same RDRC sample was dried in a hot air oven and sequentially extracted using distilled water for 18 hrs and concentrated using rotavapor. The dried extracts were stored at 4 for further uses. ℃

Phytochemical analysis
The RDRC extracts were subjected for phytochemical analysis by employing standard methods such as alkaline reagent test for flavonoids, Benedict's reagent test for carbohydrates, ferric chloride test for phenols, steroids, tannins, foam test for saponins, Mayer's test for alkaloids and Salkowski's test for glycosides (10).

Antibacterial assay
The aqueous and chloroform extracts of RDRC were checked for their antibacterial activity using the in vitro agar well-diffusion method (11). Sterile cotton swabs were used to inoculate the cultured microbial strains on Mueller-Hinton (MH) agar (HiMedia Laboratories Pvt. Ltd.) plates for antibacterial study. A sterile well-borer of 5.0 mm diameter was used to make wells, and different concentrations of aqueous and chloroform extracts were loaded (0.25, 0.50, 0.75, 1.0, 1.25 and 1.5 mg/ml). 10 mg/ml of aqueous extract was prepared in sterile distilled water, whereas 10 mg/ml of chloroform extract was prepared in Dimethyl Sulfoxide (Qualikems Fine Chem Pvt. Ltd.). Ampicillin (HiMedia Laboratories Pvt. Ltd.) was used as a positive control for an antibacterial study. Since the chloroform extract was dissolved in Dimethyl Sulfoxide (DMSO), DMSO was treated as a negative control. The bacterial plates were incubated for 24 hrs at 37 ºC. Its antibacterial activities were measured using a zone of inhibition (ZOI) in millimetre (mm). It was done in triplicates under aseptic conditions.

Determination of MIC and MBC
Minimum Inhibitory Concentration (MIC) of RDRC extract was assessed by tube dilution method. Serial two-fold dilutions of the extract were made starting from 20 mg/ml Mueller-Hinton Broth to assess bacterial growth. The concentrations used for the MIC study included 20, 10, 5, 2.5, 1.25, 0.625 and 0.3125 mg/ml. Overnight cultures of bacteria were inoculated in MH broth with different RDRC extract concentrations, incubated at 37 °C overnight in a shaker incubator (Orbital Shaker Incubator (Remi CIS-24BL), and turbidity measurements (Colorimeter, ELICO, CL137) were taken the next day at OD600. MIC was determined as the lowest concentration that inhibited the visible growth of microorganisms. In order to assess the Minimum Bactericidal Concentration (MBC), aliquots of 50 μl from all the tubes which showed no visible bacterial growth were seeded on MH agar plates and incubated for 24 hr at 37 °C. The number of colonies counted the next day. When 99.9% of the bacterial population is killed at the least concentration of an antimicrobial agent, it is fixed as its MBC. This was done by observing pre-and post-incubated agar plates for the presence or absence of bacterial colonies. Experiments were replicated thrice (n=3) and represented as mean ± SD.

Anti fungal assay
Sterile cotton swabs were used to inoculate the cultured fungal strains on Potato Dextrose Agar (HiMedia Laboratories Pvt. Ltd.) plates for antifungal study. A sterile well-borer of 5 mm diameter was used to make wells, and different concentrations of aqueous and chloroform extracts were loaded (0.25, 0.50, 0.75, 1.0, 1.25 and 1.5 mg/ml). 10 mg/ml of aqueous extract was prepared in sterile distilled water, whereas 10 mg/ml of chloroform extract was prepared in DMSO. Sporanox (Johnson & Johnson Pvt. Ltd.) was used as a positive control for antifungal study (12). DMSO was used as a negative control. The fungal plates were kept at room temperature for 5-7 days. Its antifungal activities were assessed by measuring the zone of inhibition (ZOI) in millimetre (mm). It was also done in triplicates under aseptic conditions.

Statistical analysis
All the values were expressed as mean ± S.D of three parallel measurements. Statistical analyses for the antimicrobial activities were performed using the SPSS software package. The mean values were analyzed by one-way ANOVA. To determine the statistical significance of antimicrobial activity, Duncan's Multiple Range Test (DMRT) was used. pvalues < 0.05 were regarded as significant.

Results and Discussion
Natural medicines derived from parts of plants, animals and minerals obtained from Earth have been found to play important roles in fighting various diseases. With its long history of more than 2000 years, Traditional Tibetan Medicine (TTM) has been well recognized in Tibet and other parts of the world. TTM has been considered to play a significant role in the prevention and treatment of various diseases, including cancer (5). In the current study, the extract of the pill RDRC was prepared using chloroform and water. Water was used for extraction of the polar and semi-polar constituents. However, for lipophilic compounds, lipophilic solvents such as chloroform were used for extraction. The phytochemical analysis of both extracts as shown in Table 1 indicated the presence of alkaloids, carbohydrates, flavonoids, glycosides, phenols, saponins, steroids and tannins. These RDRC extracts were screened in vitro for antibacterial activity by the agar well diffusion method (Table 2 and 3). Both the extracts did not show any activity against tested Gram-positive bacteria, as shown in Table 3. However, chloroform extract exhibited considerable activity (Fig. 1) against three Vibrio species: V. harveyi at 1, 1.25 and 1.5 mg/ ml showed zone of inhibition (ZOI) of 7.0 ± 0.0, 9.0 ± 1.0 and 10.0 ± 0.0 mm respectively; V. vulnificus at 0.75, 1, 1.25 and 1.5 mg/ml exhibited ZOI of 5.6 ± 0.5, 7.6 ± 0.5, 9.3 ± 0.5 and 10 ± 1.0 mm; V. parahaemolyticus was more sensitive with its ZOI noticeable even at 0.25 mg/ml as displayed in Table 2. Since the chloroform extract was dissolved using DMSO and diluted using distilled water, DMSO (Solvent) inhibition was also checked, and it exhibited no inhibition. RDRC extracts were assayed for antifungal activity against two fungal strains, but both the extracts were inactive and exhibited no fungicidal activities (Table 4).
MIC values were assessed for the three Vibrio species for which RDRC extract showed antibacterial activity as evidenced by the zone of inhibition. The least MIC value (3.33 ± 1.44 mg/ml) was observed for V. parahaemolyticus and the highest (5.00 ± 0 mg/ml) for V. harveyi. MBC values were calculated for V. parahaemolyticus, V. vulnificus and V. harveyi (Table  5), in which V. parahaemolyticus had the least MBC of 8.33 ± 2.88 mg/ml, which shows that the chloroform extract of RDRC was the most efficacious against V. parahaemolyticus.
Many Vibrio species have been associated with aquatic ecosystems. V, parahaemolyticus is commonly found in rivers and estuaries. Food poisoning caused by V. parahaemolyticus usually occurs in summer. It is mostly manifested in people consuming seafood like crab, shrimp, shellfish, lobster, fish and oysters (13)(14). V. parahaemolyticus is a human pathogen and can cause acute gastroenteritis due to contaminated raw or undercooked seafood. It also can cause infections in open wounds exposed to seawater.
This Gram-negative comma-shaped bacterium is the leading cause of seafood-related gastroenteritis (15). V. parahaemolyticus is reported to cause around 35000 cases of gastroenteritis per year in the USA alone (16). Certain serogroups of bacteria causing various gastroenteritis diseases turn out to be highly virulent and can even cause pandemics. One such is the O3:K6 serogroup of V. parahaemolyticus, which emerged in India in 1996 and has since spread to other Asian countries (17).
Some strains of V. parahaemolyticus have been found to be antibiotic-resistant. Hence, the use of synthetic antibiotics should be done with caution as these inhibitors based on chemical molecules often lead to bacterial drug resistance or leave residues in the environment, which can pose a threat to animal and human health (18). In this scenario, the importance of natural products to combat pathogens can be looked at. Most of the ingredients of RDRC are obtained from nature. The current study showed significant anti-Vibrio activity against V. parahaemolyticus as evidenced by the zone of inhibition of about 13 mm. In another study, natural products like cumin have been reported to have antibacterial effects on V. parahaemolyticus (19).
Vibrio vulnificus causes both foodborne and wound infections and is notorious for being responsible for the highest death rate caused by any foodborne disease agent in the USA (20). This pathogen is highly invasive and can cause septicemia in persons with reduced immunity; and is responsible for 95% of all seafood-related deaths in the USA, with a mortality rate of 60% (21). But not all strains of V. vulnificus have been found to be highly virulent (22). Most of the deaths occur within 72 hrs if proper treatment is not given. Generally, a combination of tetracyclines and third-generation cephalosporins are given as treatment. There has been increasing evidence of V. vulnificus becoming resistant to antibiotics and hence, it becomes necessary to explore newer anti-Vibrio compounds, especially of natural origin. A previous report has depicted the anti-Vibrio activity of the plant Ocimum gratissimum Linn. (23). Present study has yielded a positive result for antibacterial activity of RDRC extract against V. vulnificus, as the zone of inhibition was found to be about 10 mm. Hence, this Tibetan Precious pill, RDRC can be thought of as a part of the anti-Vibrio therapy for manifestations of V. vulnificus infections which are life-threatening.
The third bacterium we tested using RDRC extract was V. harveyi. It is facultatively anaerobic, Table 2. Antibacterial activity of RDRC extracts in comparison with standard antibiotic ampicillin against Gram-negative bacterial strains

Compound
Conc. (mg/ml)  halophilic, bioluminescent and motile (24). As with other Vibrio species, V. harveyi is also found in tropical marine waters and also lives as a commensal in the gut microflora of marine animals. V. harveyi has been found to cause mass mortality events in benthic invertebrates in the Mediterranean Sea. This pathogen has also been pinpointed as a major threat to the survival of marine animals due to disease outbreaks owing to dramatic climate change events (25). The first report giving evidence of V. harveyi being a human pathogen was published in 1989, of an 11 year old girl bitten by a shark in California and subsequently getting wound infection in the year 1985 (26). The organism was confirmed to be V. harveyi by the Centre for Disease Control, CDC.

Escherichia coli Pseudomonas aeruginosa Vibrio vulnificus Vibrio harveyi Vibrio parahaemolyticus
Another four more cases were reported later, pointing to the fact that V. harveyi can cause human infections, though the chances are rare. There have been previous reports of Silver nanoparticles of tea leaf extract (27) and colloidal Silver nanoparticles (28) offering antibacterial activity against V. harveyi. Present study also has revealed potential antibacterial activity against V. harveyi by the chloroform extract of RDRC.
The anti-Vibrio effects of RDRC could be due to the combined mode of action of different plant extracts present in the pill. Methyl gallate, one of the active ingredients of T. chebula Retz., a major component of RDRC has been found to act as an effective agent for the treatment of severe secretory and inflammatory diarrheal diseases caused by multidrug-resistant strains of V. cholerae (29). A high level of antibacterial activity was reported for V. parahaemolyticus by the use of extracts of Phytolacca americana Linn. (30). Myrtiscin and Eugenol found in Myristica fragrans Houtt., another predominant constituent of RDRC, have been shown to have excellent antibacterial action against V. cholerae (31). Our preliminary antibacterial studies for the three highly pathogenic Vibrio species have depicted the potential antagonistic action of the extract of the Tibetan precious pill RDRC against these bacteria causing life-threatening infections.

Conclusion
The current study was successful in validating the antibacterial effect of the chloroform extract of the Tibetan Precious Pill, Rinchen Drangjor Rilnag Chenmo (RDRC), against three highly infectious and virulent Vibrio species. Further studies are being planned to investigate the molecular mechanism of disruption of bacterial growth. This will definitely be useful in proving the projected success of such pills in treating a multitude of diseases ailing humankind. Globally, different ethnic groups have evolved their own traditional medical practices, some of which have really been shown to be efficient in curing dreaded diseases. With the advancements made in biochemistry and molecular biology, more scientific validation of the medicinal effects of such traditional pills is indeed the need of the hour.