In vitro Antioxidant Potential and Anti-bacterial Activities of Rajanyadi Churna Extracts Against Escherichia coli

 

Rupali Deshpande¹, Chandrashekar K.S.¹, Vasudev R Pai¹,

Venkatesh Kamath², Aswatha Ram H.N.¹*

¹Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences,

Manipal Academy of Higher Education, Manipal, Karnataka, India.

²Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences,

Manipal Academy of Higher Education, Manipal, Karnataka, India.

 

ABSTRACT:

Rajanyadi churna (RC) is a polyherbal traditional formulation prescribed in Ayurveda for treating diarrhea, gastric disorders, asthma, fever, anemia and jaundice.  In the current experiment, various in vitro models of antioxidant activity, and antibacterial activitity against Escherichia coli of Rajanyadi churna alcoholic extract (RCAL) and Rajanyadi churna aqueous extract (RCAQ) are presented.  In vitro antioxidant potential was determined for both the extracts in various models like ABTS, Nitric Oxide, DPPH, Superoxide and Hydrogen peroxide scavenging assays. The minimum inhibitory concentration (MIC) of RC extracts against Escherichia coli, Gram negative bacteria were determined using the micro-dilution technique (ATCC 11775). Our study showed a remarkable improvement in the antioxidant activity of RCAL and RCAQ in all the in vitro models.  The percentage of inhibition for RCAL and RCAQ were found to be 31% and 25% respectively against E. coli.  From this experimental study, we conclude that Rajanyadi churna extracts showed good antioxidant activity and antibacterial activity against E. coli.

 

 

INTRODUCTION:

The electrons usually move around the nucleus in pairs and when an electron dissociates from the pair, the atom or molecule becomes highly unstable which is called as a ‘free radical’¹.  The cells are thought to be damaged by the excess oxygen radical species production viz hydrogen peroxide, superoxide anion radical, and the radical hydroxyl ion². Antioxidant molecules are known to inhibit or at least retard the process of oxidation by preventing the onset and oxidizing free radical chain reactions propagation³.  Antioxidants from synthetic sources have suffered with serious adverse reactions such as carcinogenicity^4&5. The natural antioxidants on the other hand have proven to be safe, and are known to protect certain components of cell from oxidative damage caused by the oxidative species such as ROS⁶. 

 

In the recent years, the importance of research in the plant based antioxidants and their exploitation greatly increased⁷.

 

Rajanyadi churna (RC) is an Ayurvedic polyherbal classical formulation traditionally used to treat diarrhea, digestive disorders, asthma, anemia fever, and             jaundice ⁸. The role of free radicals is linked to all these diseases as reported by many authors.  The oxygen free radicals significantly contribute to tissue damage and results in various diseases and disorders of the GIT⁹. An effort has been put to establish the role of oxidative stress role in diarrheal diseases¹⁰ and, in acute rheumatic fever¹¹. Oxidative stress has been very well illustrated in, liver damage¹², pathogenesis of Fanconi's anemia¹³, and in asthma¹⁴. Diarrhea is one of the most common ailment especially among children killing more than 760000 kids every year around the world according to WHO. It is caused by the contamination of food and water with organisms like Salmonella typhi, Escherichia coli, Giardia intestinalis and Cryptosporidium parvum¹⁵.  RC has been used in the treatment of diarrhea, and therefore its activity against causative organism was carried out. Further Helander et al (1998)¹⁶ showed the antibacterial activity of natural antioxidants in vitro assay against the species of E. coli 0157:H7. Thus the present work was devised to carry out antioxidant and antibacterial activities. 

 

MATERIAL AND METHODS:

Plant materials:

The crude drugs obtained Udupi local market, India, and authenticated by botanist Usha Rani S. Suvarna, Associate Professor and HOD of Botany, M.G.M. College, Udupi.  For future reference, a voucher specimen of obtained crude drugs (PP 532A, PP 577A, PP 588, PP 593) was stored. 

 

Preparation of In-house churna:

The In-house RC was prepared in three separate batches using the method described in AFI. To obtain the required churna, the crude drugs were pulverised, passed via sieve 80#, and finally mixed in various proportions as specified in Table 1.

 

Table 1: Ingredients of Rajanyadi churna

Sanskrit name	Botanical name	Part used
Rajani (haridra)	Curcuma longa	Rhizome
Daru (devadaru)	Cedrus deodara	Heart wood
Sarala	Pinus roxhburgii	Heart wood
Sreyasi (gajapippali)	Scindapsus officinalis	Fruit
Brhati	Solanum indicum	Plant (whole)
Prsniparni	Uraria picta	Plant (whole)
Kantakari	Solanum xanthocarpum	Plant (whole)
Satahva	Anethum sowa	Fruit

 

Preparation of extracts:

Thirty g of RC extracted with ethanol in a soxhlet extractor to give alcoholic extract (RCAL).  The ethanolic extract was dried and the yield was 11.16%.  Rajanyadi churna aqueous extract (RCAQ) was obtained by separately extracting 30g of RC using distilled water by maceration and dried extract yield was 15.32%.  Both the extracts were desiccated till use.

 

Chemicals:

Sigma Chemicals were 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) and 1,1-Diphenyl-2-picryl-hydrazyl. All other chemicals of analytical grade were used.

 

DPPH (Radical) Scavenging Assay:

The DPPH assay was carried out using ethanolic solution in the concentration of 200 µM DPPH and different concentrations of RCAL and RCAQ in ethanol. 0.05 ml of each of ethanolic RCAL and RCAQ was put at concentrations (10-100 µg/ml). A control with equivolume of ethanol was prepared and UV absorbance was read out and noted after 20 min of incubation of all solutions at 517 nm. Equation below gave percentage inhibition¹⁷.

­­­­­­­­­                         (Control–test)

% Inhibition = ------------------------× 100

                              Control

 

ABTS (Radical) Cation Depolarization Assay:

When 7mM solution of ABTS was made to react with 2.45 mM ammonium persulfate, ABTS cations (ABTS⁺) were expressed.  The cationic solution was incubated for 12-16 hr in a dark chamber at normal room temperature and then used.  ABTS (0.3 ml) solution was taken and added with various amounts of RCAL (10-140 µg/ml) and RCAQ (0.5 ml).  The final volume 1 ml of tests and standard was made with ethanol. At 745 nm, UV absorbance all the solutions were read and the % inhibition were computed by using the cited formula¹⁸.

 

Nitric Oxide (Radical) Scavenging Assay:

Griess' reaction was used to generate free radicals of nitric oxide.  The incubation of standard phosphate buffer solution containing Sodium nitroprusside (5 mM) was carried out with varied amounts (25-400 µg/mL) of RCAL and RCAQ.   The extracts added to phosphate buffer (0.025 M; pH: 7.4) for 5 h at 25°C was also incubated.  Control with equivalent amounts of buffer was used for comparison.  After 5 h of incubation, dilution of the solutions were made with Griess’, reagent (0.5 mL).  The absorbance value of the chromphore formed at the time of diazotization reaction was analyzed at 546 nm¹⁹.

 

Superoxide (Radical) Scavenging Assay:

In order to scavenge potassium superoxide, the alkaline DMSO method was used. The dried DMSO and potassium superoxide was incubated together for 1 day before resulting solution was filtered and used. The aqueous solution (2.8 mL) comprising of NBT (56 µM), Ethylenediaminetetra acetic acid (10 µM) and 10 mM of potassium phosphate buffer was added to 200 µL of filtrate.  Extracts and the standard were added at various concentrations (20-140 g/mL). At 560 nm, the absorbance was measured with pure DMSO as a control²⁰.

 

Hydrogen peroxide scavenging assay:

pH 7.4 phosphate buffer solution containing Hydrogen peroxide strength of 40 mM was prepared.  One hundred μg of extracts per mL in water, distilled was combined with 0.6 mL of 40mM strength of Hydrogen peroxide.  After 10 min, analysis was carried out using UV spectrophotmeter at 230 nm against the blank, depriving H₂O₂. Percentage of H₂O₂ scavenged by both the extract samples and reference standard were computed²¹.

 

                                       (A₀ – A₁)

% Scavenged [H₂O₂] = ------------ x100

                                              A₀

Statistical Analysis:

The data was mentioned as mean ± standard of triplicate determinations.  IC₅₀ values were analyzed for Linear regression.

 

In vitro assay for antibacterial activity:

In vitro antibacterial assay was performed by micro-dilution method (Eloff, 1998) using 96-well microtitre plates for the determination of MIC values of RC extracts against bacterial species (Gram-negative) like Escherichia coli (ATCC 11775).  Healthy colonies of the bacterial culture were transferred into 10 ml Mueller-Hinton (MH) broth to E. coli stock in a tube.  Overnight growth was done with shaking at 37⁰C.  The turbidity was adjusted using MH broth for the E. coli culture.  McFarland standard, having 10⁸ colony forming units/ml (CFU/ml) was matched with E. coli.  A concentration of 50 mg/ml of dried extract of RC was added to 70% ethanol while aqueous extract was added to sterile water.  Serial dilution of extracts in 96-well microtitre plate was carried out using sterile water (100µg/ml).  Neomycin (100µl) a similar two-fold micro dilution was used as a positive control.  Solvent used was 70% ethanol, negative control was water.  Each well was added with bacterial culture (100µl).  The microtitre plates were covered with para-film were incubated for about 24 h at 37⁰C.  Absorbance was recorded using UV spectrometer and the percentage inhibition was calculated ²².

 

Total Phenolic Content (TPC):

The Folin-Ciocalteau method was used to calculate TPC of both RC extracts. 0.5 mL of extract at concentrations of (125g, 250g, 500g, 1000 g), 2.5 mL Folin-Ciocalteau reagent (1/10 diluted), and 2 mL, 7.5 percent sodium carbonate (w/v) was added, and allowed to stand for 30 minutes while being shaken intermittently, absorbance (blue colour) was analysed at 765 nm. Sodium carbonate solution served as the control (2mL of 7.5 percent sodium carbonate dissolved in 2.55 mL of distilled water). Total phenolic content was reported as gallic acid equivalents (GAE) in g/ml of dry extract.²³.

 

Total Flavonoid Content (TFC):

Five ml of aluminium trichloride (2%) was mixed with same amount of sample (1 mg/mL).  After 10 minutes, absorbance at 415 nm was taken against a blank, i.e. 5 ml of sample with 5 ml methanol without aluminium trichloride.  The standard graph of quercetin in concentration of 10–100 µg/ml was used to analyse the total flavonoid content.  Average of three readings was considered and quercetin equivalents (QE) was expressed based on a dry weight basis²⁴.

 

RESULTS:

In vitro antioxidant assays carried out for RCAL and RCAQ showed significant scavenging activity against DPPH, ABTS, nitric oxide generated free radicals, superoxide dismutase and in Hydrogen peroxide assay, were of concentration dependent for the given concentrations of experimental models. In an exceptional case, where RCAQ showed more activity than RCAL.  The percentage scavenging for various concentrations of both RC extracts and standard ascorbic acid equivalence are depicted in [Fig. 1-5].

 

 

Figure 1. Percentage scavenging of DPPH radical by extracts (values are mean ± SD and n = 3.)

 

Figure 2. Percentage scavenginf of ABTS radical by extracts (values are mean ± SD and n = 3.)

 

Figure 3. Percentage scavenging of NO radical by extracts (values are mean ± SD and n = 3.)

 

Figure 4. Percentage scavenging of Superoxide radical by extracts (values are mean ± SD and n = 3.)

 

Figure 5. Percentage scavenging of H₂O₂ radical by extracts (values are mean ± SD and n = 3.)

 

IC₅₀ values was determined for the various models and is presented in Table 2. RCAL showed significant effective scavenging for various assays in comparison to RCAQ.  The effectiveness of RCAL in scavenging of the free radicals is given in the sequence of, Superoxide>ABTS>H₂O₂>DPPH>NO.  On comparative basis, maximum activity was observed in inhibiting the generation of superoxide and the least was observed in Nitric oxide assay.  RCAQ showed maximum activity in comparison with other models, NO> Superoxide>ABTS> H₂O₂>DPPH.

 

Table 2: IC₅₀ values of Standard, RCAL and RCAQ

IC50 Values
Sample	ABTS Assay	DPPH Assay	H2O2 Assay	Nitric oxide Assay	Superoxide Assay
Standard	22.19	8.41	65.36	8.16	21.86
RCAL	18.17	38.86	19.10	169.01	10.38
RCAQ	42.09	144.02	130.32	14.73	23.57

 

In vitro antibacterial assay:

Assay was performed with E. coil as it is a very important causative organism for diarrhea. Dilution method was followed and the percentage of inhibition for RCAL and RCAQ was found to 31% and 25% respectively.

 

TPC:

Total phenol content of RCAL and RCAQ of was analysed to be 234.112 and 473.128 mg/g respectively when compared with Gallic acid.

 

TFC:

Total flavonoid content of RCAL and RCAQ were 110.81 mg/g and 117.72 mg/g respectively when compared with Quercetin.

 

DISCUSSION:

The antioxidant activity of DPPH depends on the capacity of DPPH free radical to undergo reduction.  RC extracts form the corresponding hydrazine from the radical after reduction when it reacted with donors of hydrogen in RC extract anti-oxidant principles²⁵. ABTS radical cation shows characteristic wavelength absorption. The ability to inhibit the absorbing species of ABTS is used for analysis.  RC extracts found to show activity by scavenging or inhibition of the reactive species and acts as an antioxidant towards ABTS free radical²⁵.

 

Stable nitrates and nitrites via intermediates such as NO₂, N₂O₄ and N₃O₄ are formed as a result of reaction between nitric oxide unstable radical with oxygen (Griess reagent). Sodium nitroprusside in standard phosphate saline was incubated at 25⁰C, resulted in the formation of nitrite which was reduced by RC extracts competitively reacts with the NO, thereby inhibiting nitrite generation²⁶. After taking oxygen into the live cells, superoxide anion is generated and considered to be the strongest reactive oxygen species.  Harmful Reactive Oxygen species and the free radicals viz hydroxyl and also hydrogen peroxide, these are the products of Superoxide anion, which induce oxidative damage²⁷.  Oxygen and hydrogen peroxide are the catalyzed products of dismutation of superoxide anion by the superoxide dismutase enzyme. The first decrease in product of oxygen is superoxide anion, measured as inhibition of generation of 0^2•28.  The extracts showed inhibition of superoxide generated in the reaction mixture result obtained was depedent on concentration.

Hydrogen peroxide is a weak oxidizing agent, still readily react and crosses biological membranes. The formation of hydroxyl radical is from H₂O₂, and is responsible for cytotoxicity. Removing H₂O₂ is essential to protect living systems²⁹ from harmful effects of it.  RC extracts scavenged H₂O₂, which can be accredited to phenolic groups and could donate electrons to hydrogen peroxidase, hence neutralizes to water³⁰.

 

Gram-negative bacteria have been tough organism to deal with mostly due to the impermeability of it membrane for antibacterial compounds. Such impermeability could be due to the presence of LPS (lipopolysaccharides) in bacterial cell outer membrane^31&32.  The herbal extracts may also succumb to the impermeability of cell membrane and possibly show low percentage of inhibitory activity. Anti-bacterial activity could be due to presence of phenolic compounds^33,34.  Flavonoids are the chief among various categories of phenolic compounds of the plants. The oxidizing molecules are at most, including singlet oxygen and many free radicals involved to cause several diseases are scavenged effectively by Flavonoids³⁵.

 

CONCLUSION:

From this study, it can be decided that Rajanyadi churna extracts showed significant antioxidant activity in all in vitro models.  This could be due to the phenolic component and flavonoid content of the churna extracts.  Since free radicals are linked to many diseases, it is easier to assume that the churna works through its antioxidant mechanism also.  Further research is important to elucidate the actual mechanism to correlate for treatmenting the diseases at the cellular or molecular level.

 

ACKNOWLEDGEMENT:

The authors sincerely thank Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, India for providing the necessary facilities to carry out this work.

 

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Accepted on 09.06.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(11):5120-5125.