INTRODUCTION:

Generally functional foods means despite tremendous strides in modern medicine, prevention and treatment of chronic ailments with physiologically active food components or beverages as well as dietary supplements is of revived interest in the present in todays health conscious world. The concept of applying functional foods and dietary supplements as adjunct therapy dates back 2,500 years ago when Hippocrates, the father of modern medicine says that “Let food be thy medicine and medicine be thy food.” Tulsi in Hindi or Tulasi in Sanskrit (holy basil in English) is a highly revered culinary and medicinal aromatic herb from the family Lamiaceae that is indigenous to the Indian subcontinent and been used within Ayurvedic medicine more than 3000 years. In the Ayurveda system tulsi is often referred to as an “Elixir of Life” for its healing powers and has been known to treat many different common health conditions. In the Indian Materia Medica tulsi leaf extracts are described for treatment of bronchitis, rheumatism, and pyrexia and any bacterial infection Other reported therapeutic uses include treatment of epilepsy, asthma or dyspnea, hiccups, cough, skin and haematological diseases, parasitic infections, neuralgia, headache, wounds, astringent and inflammation and oral conditions. The juice of the leaves has been applied as a drop for earache, while the tea infusion has been used for treatment of gastric and hepatic disorders. The roots and stems were also traditionally used to treat mosquito and snake bites and for malaria. There are three types of tulsi are commonly described. Ocimum tenuiflorum (or Ocimum sanctum L.) includes 2 botanically and phytochemically distinct cultivars that include Rama or Sri tulsi (green leaves) and Krishna or Shyama tulsi (purplish leaves) while Ocimum gratissimum is a third type of tulsi known as Vana or wild/forest tulsi (dark green leaves). The different tulsi types exhibit great diversity in morphology and phytochemical composition including secondary metabolites, yet they can be distinguished from other Ocimum species by the colour of their yellow pollen, high levels of eugenol, and smaller chromosome number. Despite being distinct species with Ocimum tenuiflorum having six times less DNA than Ocimum gratissimum they are traditionally used in the same way to treat similar ailments. For consistency, this review uses the term tulsito refer to both Ocimum tenuiflorum or Ocimum gratissimum.

MATERIAL AND METHODS:

Tulsi leaves were obtained from the medicinal garden of pravara rural college of pharmacy, Pravara nagar. Leaves were separated from the stem then washed in clear water and dried until they were adequately dry to be ground (dried for 7 days). Dried leaves were powdered separately in an electric grinder until a dry andhomogenous powder was obtained. Ethanolic, Hexane, Chloroform extract was prepared from the powder obtained using “cold extraction method

300 grams of finely powdered Ocimum sanctum (Linn.) was macerated with Tulsi (Ocimum santum) extract preparation; (a) Ocimum sanctum plant; (b) leaves separated and dried; (c) leaves ground to powder; (d) extract obtained 100% ethanol and other solvent. It was then subjected to filtration with Whatman filter paper to obtain a clear filtrate. The filtrate so obtained was reduced at a low temperature of less than 60⁰C to obtain a solid residue of Ocimum sanctum (Linn.) Extract From 300 grams of Ocimum sanctum (Linn.) powder dissolved in 1 liter of ethanol and other solvent, 18 gram of extract (residue) was obtained and thus the yield was 6% w/v. Accurately weigh 1gm of each extract was reconstituted in 10 ml of respective solvents to obtain stoke solution.

Further the dilutions were made with respective solvents. Accurately weigh of 10 mg of Standard Gentamycin was dissolved in 10 ml of distilled water to get 1mg/ml. The different dilutions and standard pipetted out on into the marked plates. These were left for incubation at 37°C for 24 hrs and 25º C for 36 hrs. After incubation zones of inhibition were measured (from antibiotic zone measurement scale) in mm and compared with standard.

For the extraction of curcumin from turmeric crude powder microwave extraction method use in that for 0.5 g of turmeric powder was weighed and dissolved in 10 ml acetone and put in microwave chamber. Acetone which was used as extraction solvent has good dissipation factor (tan δ = 0.5555) which can be heated up to high extent and dissipate the microwave energy. Extraction was carried out at different microwave operating powers varied between 100-450 W and different irradiation times of 0.5-3 min. The samples were subjected to microwave irradiation in an intermittent way of irradiation cooling irradiation for extraction time of up to 3 min, because longer irradiation time and higher power caused boiling of solvent. After that, the solvent was separated through 0.45 μm filter and evaporated under vacuum, the residue was weighed and dissolved in 10 ml methanol for HPLC analysis for concentration of curcumin.

Fig. No 1 Leaves and extract of tulsi

Collection of test organism:

Collection of test organism and preparation of stock culture: The following strains were obtained for the antimicrobial tests analysis. Gram positive bacteria were Staphylococcus aureus. All the microbial strains were obtained from microbial department of Padmashri Vitthalrao Vikhe Patil College, Pravaranagar. Nutrient broth medium was prepared and autoclaved. After the bacterial cultures were inoculated to separate flasks and incubated in shaker for 24 hours

Microbiological Assay:

The test organisms included for study were gram positive Staphylococcus aureus in detail.

Preparation of Media:

For 100 ml of media,40 gm of muller-hinton agar is dissolved in 100 ml distilled water. 250 ml media prepared and autoclaved at 121°C to 15-20 min at 15 lbs/inch².

Preparation of Disk:

Freshly prepared and sterilized molten media was poured onto Petri plates inside Laminar and after pouring UV light turned on to avoid contamination on plates while media solidifying. It was left for half an hour for proper solidification. After media gets solidified than UV light is turned off and 10μl of bacterial suspension pipetted into plates and swabbed.

Fig 2 Zone of inhibition

Sterile discs were put (with the help of forceps) on plates along with one disc of standard (6 disk was put on a plate).

Observations:

Table- Tulsi leaves serial dilution showing the Zone of inhibition (in mm) against Staphylococcus aureus (Gram positive bacteria)

Extract	Dilution 1	Dilution 2	Dilution 3	Dilution 4	Dilution 5	Dilution 6	Gentamycin
Chloroform extract	11	11	10	9	5	4	17
Hexene Extract	9	7	7	4	3	2	17
Alcoholic Extract	21	21	21	21	21	21	17

Fig 03 Tulsi leaves serial dilution showing the Zone of inhibition (in mm) against Staphylococcus aureus (Gram positive bacteria).

Instruments used in formulation:

Electronic balance Bulk density apparatus, Standard sieve 30#, Hot air oven, Tablet compression machine, Friability apparatus, Hardness tester (Monsanto, India); Vernier califer, USP Type I tablet dissolution apparatus ,Infrared spectroscopy

Formulation of herbal API loaded solid dosage forms:

Pre formulation studies:

Pre formulation studies are done where physical, chemical and mechanical properties of a new drug substance or chemical entities are characterized alone and on combining with excipients in order to develop stable, safe and effective dosage form.

Tulsi and termaric extract -polymer compatibility studies:

Compatibility amongst both extract and the excipients used in the formulations were studied by FTIR analysis. An IR spectrum properly blended mixtures of extract with the excipients were recorded in FTIR spectrophotometer in the scanning range of 1600-15850 and1500-1400 cm^-1with a resolution of 4cm^-1 The basic purpose was to observe any changes in the spectrum pattern of the extract due to polymers and thus identify the chances of any chemical interactions.

Pre compressional studies:

1) The flow properties and compressibility of extract and excipient powder blends for the purpose of tablet were evaluated by measuring Angle of Repose (fixed funnel method)

2) Bulk Density (BD) and Tapped Bulk Density (TBD) by Cylinder method on Bulk and tapped density apparatus

3) Carr‟s Compressibility Index using the equation: Carr‟s Compressibility Index (%) = [(TBD-BD)/ TBD] x100

4) Hausner‟s ratio was determined by the equation: Hausner‟s Ratio = TBD/ LBD.

Hausner‟s ratio less than 1.25 indicates good flow while greater than 1.5 indicates poor flow using standard procedures The values obtained after testing are compared with the standard values and inferences were drawn In Table 2

Procedure for formulation:

Herbal tablets were prepared by wet granulation, the wet dough mass of all well mixed ingredients were passed through sieve no. 16 so as to get uniform sized granules. After 3-4hrs of air drying the granules were further dried in hot air oven for 20-30min at 45⁰C-50˚C. Dried granules were further sieved and then magnesium stearate and Talc were added as lubricants Next tablets were subjected to direct compression using single punch tablet machines (P.R.C.O.P LONI) Hardness of the tablets was maintained about 4-5 kg/cm. The all ingredient are contain in table no 1

Quality control Test:

Tulsi and Turmeric herbal combination tablets was prepared evaluated they are as follows

1) Hardnesswas determined using Monsanto hardness tester

2) Friability was determined using Roche Friabilator

3) Thickness and diameter of the tablets were determined using Vernier calipers

4) Weight variation test was carried out as per official methods with the specification limit that not more than two of the individual weight deviates from the average weight by 10 % and none should deviate by more than twice that percentage.

5) Disintegration Test using USP-DT apparatus

The observation of these all quality control parameter of herbal tablet are given in table no 3.

Table- 1: Formula for herbal antimicrobial tablet (per 100gm) in gm

Ingredient	Formula 1	Formula 2	Formula 3	Formula 4
Tulsi and turmeric extract	3.50	4.00	4.50	5.00
Chitosan	22.5	22.5	22.5	22.5
Lactose	65.8	44	12.3	14.2
Talc	2.5	3.5	5.5	2.5
Magnesium Stearate	1.5	3.5	6	1.5
Colloidal Silica/Aerosil	1.2	2.2	4	1.2
PVP	2.5	5	7.5	2.5

Table- 2: Pre compressional evaluation of the powder blend

Formulations	Angle of repose	Bulk Density	Tapped Density	Compressibility Index (%)	Hausner‟s Ratio
Formulation 1	24.56±0.695	0.740±0.002	0.881±0.001	13.521±0.008	1.182±0.002
Formulation 2	22.96±1.211	0.729±0.003	0.879±0.003	15.781±0.003	1.198±0.003
Formulation 3	24.78±0.473	0.702±0.009	0.809±0.009	13.928±0.007	1.189±0.002
Formulation 4	23.84±0.512	0.719±0.001	0.821±0.003	15.127±0.002	1.151±0.004

Table- 3: Post compression evaluation of herbal antimicrobial tablets

formulations	Hardness	Friability	Diameter	Thickness	Weight variation	Disintegration
Formulation 1	4.27±0.32	4.27±0.32	7.49±0.002	4.62±0.005	226.74±0.51	45.00±1.0
Formulation 2	4.31±0.17	4.31±0.17	7.37±0.001	4.53±0.002	226.21±0.98	47.00±1.0
Formulation 3	4.33±0.21	4.33±0.21	7.67±0.003	4.64±0.003	226.84±0.52	45.00±1.0
Formulation 4	4.27±0.26	4.27±0.26	7.54±0.006	4.61±0.001	226.16±0.76	46.00±1.0

RESULT:

The present study clearly indicates that Ocimum sanctum is a rich source of phyto-chemical constituents. The antimicrobial efficacy of Ocimum sanctum leaves indicates that the plant possesses potent antimicrobial properties as well as Ocimum is widespread in India, it can be recommended as an easily available and renewal source of antimicrobial agent and curcuma longa containing curcumin show antimicrobial and wound healing activity instead of synthetic chemicals.

CONCLUSION:

Antibacterial activity of different Ocimum sanctum extracts against Kleibsiella pneumonia (Gram positive bacteria) and Staphylococcus aureus (Gram negative bacteria) were studied. According to the results, all different types of extracts obtained from Ocimum sanctum leaves shown to be with antibacterial activity against tested microbial pathogens. The herbal formulation of tablet with combination of two extract, having great activity of antimicrobial, antifungal, anti-inflammatory and wound healing compare to synthetic chemicals and this formulation we can use in various diseases without any side effect.

REFERENCES:

1. Mitra A, Dewanjee D, Dey B. Mechanistic studies of lifestyle interventions in Type 2 diabetes. World J Diabetes. 3(12), 2012, 201-207.

2. Mitra A. Effects of a Composite of Tulsi Leaves, Amla, Bitter Gourd, Gurmur Leaves, Jamun Fruit and Seed in Type 2 Diabetic Patients. J Clin Diagn Res. 6, 2007, 511-520.

3. De B, Bhandari K, Singla RK, Katakam P, Samanta T, Kushwaha DK, Gundamaraju R, Mitra A. Chemometrics optimized extraction procedures, phytosynergistic blending and in vitro screening of natural enzyme inhibitors amongst leaves of Tulsi, Banyan and Jamun. Pharmacog Mag. 11(44), 2015, 522-532.

4. Sen G, Bera B. Black tea as a part of daily diet: A boon for healthy living. IJTS. 9(2–3), 2013, 51-59.

5. Bhandari K, De B, Katakam P, Adiki SK, Mitra A. Development, quality control of CTC black tea solid dosage formulations and in vivo evaluations of its AChE inhibitory effects. IJOPILS. 5(1), 2016, 9-34.

6. Ghosh Hajra N, Yang CWM. Diversification of the Tea Products - Global Scenario. J Tea Sci Res. 5(3), 2015, 1-10.

7. Radhika PR, Karanam D, Sivakumar T. Fabrication of sustained release matrix tablets of venlafaxine hydrochloride using eudragits. IJRAP. 2, 2011, 1386-1389.

8. Prakash Katakam Formulation and quality evaluations of tulsi tea tablets - a tea diversification product Baishakhi De et al., Int. J. Pharm and Ind. Res., Vol.–08 (02) 2018 [104-109]

9. Mallikarjun S, Rao A, Rajesh G, Shenoy R, Pai M. Antimicrobial efficacy of Tulsi leaf (Ocimum sanctum) extract on periodontal pathogens: An in vitro study. J Indian Soc Periodontol; 2016; 20:145-50.

10. Eswar P, Devaraj CG, Agarwal P; Anti-microbial Activity of Tulsi {Ocimum Sanctum (Linn.)} Extract on a Periodontal Pathogen in Human Dental Plaque: An Invitro Study; Journal of Clinical and Diagnostic Research. 2016; 10(3): ZC53-ZC56.

11. Ali H, Dixit S; In vitro antimicrobial activity of flavanoids of Ocimum sanctum with synergistic effect of their combined form; Asian Pacific Journal of Tropical Disease 2012; S396-S398.

12. Hammer KA, Carson CF, Riley TV, Antimicrobial activity of essential oils and other Plant extracts. J Appl Microbiol. 1999; 86:985-990.

13. Singh V, Amdekar S, Verma O, Ocimum Sanctum (tulsi): Biopharmacological activities. Web Med Cent. Pharmacolo. 2010; 1:1-7.

14. Mittal R, Kumar R, Antimicrobial activity of Ocimum sanctum leaves extracts and oil Journal of Drug Delivery and Therapeutics. 2018; 8(6):201-204

Received on 17.11.2019 Modified on 31.12.2019

Research J. Science and Tech. 2020; 12(1): 69-73.

DOI: 10.5958/2349-2988.2020.00009.1