Development of Neem and Tulsi Tablets for Cardiovascular Care

Neem, belonging to the family Meliaceous, has been traditionally employed for its antimicrobial, anti-inflammatory, and antioxidant effects. The plant contains bioactive constituents like azadirachtin, imboiled, cardiovascular diseases (CVDs) such as hypertension, atherosclerosis, myocardial infarction, and stroke are the leading causes of morbidity and mortality worldwide. The search for safe, effective, and affordable natural remedies has turned scientific. Among various medicinal plants used in traditional systems of medicine, Neem (Azadirachta indica) and Tulsi (Ocimum sanctum) have gained considerable attention due to their broad spectrum of pharmacological activities. The global view is changing towards the development and therapeutic use of safer preparations from medicinal plants for controlling various diseases. Azadirachta indica A., Juss, locally famous as “Neem”, is a popular herb for its medicinal value in a wide range of diseases including cardiovascular disorders, such as, hypertension and cardiac arrhythmia [29].

Neem (Azadirachta indica)

   ↓ [Antioxidant, Anti-inflammatory, Lipid-lowering actions]

Tulsi (Ocimum sanctum)

   ↓ [Stress modulation, HDL enhancement, Vaso protection]

Combined Effect

   → [Figure 1.3: Comparative mechanisms of Neem and Tulsi contributing to cardiovascular protection. Endothelial repair, Reduced atherogenesis, Cardio protection]a. Antihyperlipidemic Effects. [40].

1.4 Pharmacological activities of neem: [15]

Figure - 1.2 : Different Action of Neem

1.5 Cardiovascular benefits of neem :

a. Hypolipidemic and Antiatherogenic

Neem extracts have demonstrated lipid-lowering effects by reducing total cholesterol, triglycerides, and LDL while increasing HDL levels.

Mechanism: Enhances bile acid excretion and modulates hepatic lipid metabolism.

Evidence: preliminary human trials show neem leaf extract decreases serum cholesterol significantly.

b. Antioxidant and Anti-inflammatory Activity

Neem leaves contain potent antioxidants such as quercetin and nimbolide that neutralize free radicals, preventing oxidative stress—a major contributor to endothelial dysfunction and atherosclerosis.

Mechanism: Inhibition of lipid peroxidation and reduction of C-reactive protein levels.

c. Antihypertensive Activity

Studies suggest neem extracts cause vasodilation and reduce blood pressure through calcium channel blockade and nitric oxide modulation. Human relevance: Small clinical studies indicate a mild but sustained reduction in systolic and diastolic blood pressure.

d. Antithrombotic and Anti-platelet Effects

Neem may prevent platelet aggregation and thrombosis formation, reducing risk of myocardial infarction and stroke..

e. Hypoglycaemic and Insulin-Sensitizing Effects

By improving glucose metabolism, neem indirectly helps prevent diabetic cardiovascular complications.

1.6 Tulsi [Ocimum sanctum]

Plant Tulsi  (Ocimum  sanctum  Linn.)  belongs  to  the Lamiacae family which is known for its specific aromatic fragrance  and  medicinal  values  in  traditional  medicine system.  Tulsi  considered  as  the  sacred  plant  in  Indian subcontinent.  Its  medicinal  properties  have  been mentioned  in  the  ancient  medicinal  texts  of  various civilizations of the world [1]..

Pharmacological Active Components:

Tulsi contains several bioactive compounds contributing to its cardiovascular benefits:

Eugenol – an antioxidant and anti-inflammatory agent that protects cardiac tissue.

Ursolic acid – improves lipid metabolism and reduces atherosclerotic plaque formation.

Rosmarinic acid – exerts antioxidant and endothelial-protective effects.

Apigenin and Luteolin – flavonoids with potent free radical scavenging properties.

1.7 Pharmacological Activities of Tulsi :

- Cardioprotective effect, Anti-inflamatory activity ,Anti-diabetic effect ,Anti-ulcer activity ,

Anti-microbial activity , Immunomodulator effect , Hypolipidemic effect [15

METHOD AND MATERIALS

⁎ Materials used in the experiment

Table - 1 : List of chemicals

1] Collection of plant materials

In the present research work Neem and Tulsi leaves were collected from LCIT medicinal garden Bilaspur and authenticated by the head of the department of botany ,GGU, Bilaspur, Chhattisgarh, India.

⁎ Plant Profile of Neem and Tulsi

1. Neem

Botanical Name - Azadirachta indica A. Juss.

Common Names - Neem, Indian lilac, Margosa, Nim.

Family - Meliaceae

Genus - Azadirachta

Species - Azadirachta indica

Geographical Distribution: Neem is native to the Indian subcontinent and is widely cultivated in tropical and subtropical regions. It grows well in dry climates and is drought tolerant.

Botanical Description: Neem is a medium-to-large evergreen tree, commonly about 15–20 m tall. It has a straight trunk with rough grey-brown bark. The leaves are alternate and pinnately compound, bearing several narrow, serrated leaflets. Its flowers are small, white, fragrant, and arranged in axillary panicles. The fruit is an oval yellow drupe containing a single seed.

Parts Used : Leaves ,Bark, Seed ,Seed oil, Flowers, Fruits.

Major Phytoconstituents; Azadirachtin, nimbin, nimbidin, nimbolide, gedunin, quercetin, tannins, flavonoids, limonoids, and fatty acids.

Traditional Uses: Neem has been traditionally used in Ayurveda for skin disorders, dental hygiene, fever, inflammation, wound care, and infections. It is also used as a natural insecticidal plant product.

2. Tulsi

Botanical Name - Ocimum tenuiflorum L.

Synonym: Ocimum sanctum L.

Common Names - Tulsi, Holy basil, Sacred basil, Tulasi.

Family - Lamiaceae

Genus -Ocimum

Species - Ocimum tenuiflorum

Geographical Distribution: Tulsi is native to the Indian subcontinent and grows widely in tropical and subtropical Asia. It is commonly cultivated in household gardens throughout India and Southeast Asia.

Botanical Description: Tulsi is an aromatic, erect, branched herb or small subshrub, generally 30–90 cm tall. The stem is often hairy and quadrangular. Leaves are opposite, simple, ovate, serrated, and strongly aromatic. Flowers are small, white to pale purple, arranged in terminal spikes. The fruit consists of four small nutlets.

Parts Used : Leaves, Aerial parts ,Seeds ,Roots ,Essential oil

Major Phytoconstituents: Eugenol, methyl eugenol, ursolic acid, rosmarinic acid, linalool, β-caryophyllene, apigenin, orientin, vicenin, tannins, and flavonoids.

Traditional Uses: Tulsi is widely used in Ayurveda for cough, cold, fever, respiratory disorders, stress, inflammation, and digestive complaints. It is also valued as a sacred plant in India.

2]  Extraction of Neem and Tulsi using Soxhlet apparatus

Procedure :

Preparation of hydroalcoholic extract (Soxhlet Extraction)

Materials Needed:

• Neem powder (250g)
• Tulsi powder (250g)
• Ethanol (4L)
• Soxhlet apparatus (including the extraction chamber, siphon, condenser, and boiling flask)
•  Heating mantle or water bath (set to 65°C)
• Separatory funnel (for separating extracted solution, if needed)
• Filter paper (if needed for purification of extract)
• Weighing balance (for accurate measurement)

Procedure:

A. Preparation:

• 250g of Neem powder and Tulsi powder grinded them coarsely to increase the surface area for extraction.
• The extraction chamber was cleaned and properly fitted to the condenser and boiling flask.

B. Solvent Preparation:

• 2L of ethanol measured and poured it into the boiling flask of the Soxhlet apparatus. Ethanol will act as the solvent for the extraction.

C. Loading the Extractor:

• The powder (250g) into the extraction thimble (usually a cellulose thimble). This thimble will hold the plant material during the extraction process.
• Inserted the thimble containing the powder into the Soxhlet extraction chamber.

D. Assembling the Soxhlet Apparatus:

• The Soxhlet chamber connected to the condenser, ensuring that the cooling water flows properly to prevent overheating.
• The boiling flask beneath the extraction chamber was fitted.

E. Heating :

• Placed the boiling flask with ethanol on a heating mantle or water bath. Fixed the temperature to 65°C. The solvent (methanol) will begin to evaporate and condense in the Soxhlet chamber.
• The ethanol vapors were condensed into the extraction chamber, where they were dissolved the active compounds from the neem and tulsi powder. The condensed l will then siphon back into the boiling flask.

F. Extraction Process:

• The process was continued for 12 hours, during which time the methanol will repeatedly extract the compounds from the neem and tulsi powder. The cycle of boiling, condensation, and siphoning should occur continuously during this period.
• The extraction chamber was filled with solvent, and after a certain amount of time, the solvent was siphon back into the boiling flask, allowing fresh solvent to extract the neeem’s and tulsi’s active compounds.

G. Completion of Extraction:

• After 12 hours, the extraction process stopped. The methanol was have extracted the desired compounds from the neem and tulsi powder.

H. Post-Extraction:

• The boiling flask from the heating source was removed. If needed. The extract can be concentrated further by removing the solvent (methanol) under reduced pressure using a rotary evaporator or simply evaporating it under low heat if desired.

I. Final Steps:

• Once the ethanol is removed, the concentrated neem and tulsi extract was collected.

J. Preliminary phytochemical screening of HA extracts

i. Saponins Test

Saponins are glycosides with foaming properties when shaken with water. Here are the chemicals commonly used in their detection:

a) Frothing Test:

• Distilled Water: Used to extract saponins from the plant material by boiling or  soaking.
• Test: Shake the plant extract with water and observe if foam persists, indicate the presence of saponins.

b) Frothing with Alcohol:

• Ethanol or Methanol: Solvent used for extraction.
• Test: After shaking the extract with alcohol, foam formation indicates the presence of saponins.

ii. Tannins Test

Tannins are polyphenolic compounds that often form precipitates with metal salts. The following reagents are commonly used for tannin detection:

a) Ferric Chloride (FeCl₃) Test:

 • Ferric Chloride (FeCl₃): A reagent that reacts with tannins to form a blue,  green, or black color.

 • Test: Add a few drops of ferric chloride solution to the plant extract. A color change to blue, green, or black indicates the presence of tannins.

b) Lead Acetate Test:

  • Lead Acetate (Pb(C₂H₃O₂)₂): Forms a white precipitate with tannins.

  • Test: Add lead acetate solution to the plant extract. A white precipitate indicates tannins.

iii. Alkaloids Test

Alkaloids are nitrogenous organic compounds with pharmacological effects, and several reagents are used to detect them. Common tests for alkaloids include:

a) Mayer's Test:

• Mayer’s Reagent: A solution of potassium mercuric iodide (KHI).
• Test: Add Mayer's reagent to the plant extract. A creamy white  indicates the presence of alkaloids.

b) Dragendorff's Test:

• Dragendorff's Reagent: A solution of bismuth nitrate in acetic acid, usually combined with KI.
• Test: Add Dragendorff’s reagent to the extract. A bright orange or reddish- brown precipitate indicates            the presence of alkaloids.

c) Wagner's Test:

• Wagner's Reagent: A solution of iodine (I₂) in potassium iodide (KI).
• Test: Add Wagner's reagent to the plant extract. A reddish-brown precipitat  indicates alkaloids.

d) Hager's Test:

• Hager's Reagent: A solution of picric acid (C₆H₃(NH₂)O₂).
• Test: Add Hager’s reagent to the extract. A yellow precipitate indicates the  presence of alkaloids.

e) Tannic Acid Test:

• Tannic Acid: In some cases, tannic acid is used to precipitate alkaloids,  particularly in qualitative tests.
• Test: Add tannic acid to the extract. A precipitate indicates alkaloids.

f) Sulfuric Acid Test:

• Concentrated Sulfuric Acid: A strong acid that may cause alkaloids to form a colored solution.
• Test: Add concentrated sulfuric acid to the extract. If alkaloids are present, reddish, orange, or purple color may develop, depending on the alkaloid.

3] TLC of Extract

Procedure : The ethanolic extracts of neem and tulsi were analyzed by Thin Layer Chromatography (TLC) using pre-coated silica gel 60 F254 plates. Approximately 10 mg of each extract was dissolved in 1 mL methanol, and the solutions were applied as small spots on the TLC plate using a capillary tube. The plate was developed in a solvent system of Toluene : Ethyl acetate  (5:4:1, v/v) until the solvent front migrated about 8 cm. The plate was then removed, air-dried, and examined under UV light at 254 nm and 366 nm. The separated spots were observed, and the Rf values were calculated using the formula:

Rf = Distance travelled by the compound / Distance travelled by the solvent front

4] UV- Spectroscopy of Extract

Procedure :Prepare extract solution: dissolve 100 mg extract in 100 mL ethanol.Filter the solution using Whatman No. 1 filter paper.Switch on the UV–Visible spectrophotometer and allow it to warm up.Fill a quartz cuvette with methanol and set it as the blank.Scan the sample solution from 200–800 nm. Record the wavelength of maximum absorbance (λmax).Measure absorbance at the selected λmax. Use the absorbance value for estimation or comparison of the extract.Common Wavelength Range Extract Approximate λmax range, Neem extract 200–800 nm , Tulsi extract 200–800 nm.

5] Selection and Compatibility of Excipients :

Procedure :Take equal quantities of Neem extract, Tulsi extract, and selected excipient.Mix each combination separately in small airtight vials.Keep one set at room temperature and one set at accelerated condition, such as 40 ± 2°C and 75 ± 5%  relative humidity, for about 2–4 weeks.Observe for: Change in colour, Change in odour ,Caking or liquefaction ,Moisture absorption, Change in flow property ,Change in tablet hardness or friability ,Compare the mixtures with freshly prepared samples.

Compatibility of Neem and Tulsi Extracts with Excipients :

Neem extract + Lactose - Compatible when extract is properly dried.

Tulsi extract + Lactose - Compatible when protected from moisture.

Neem/Tulsi extract + DCP - Compatible in dry tablet formulation.

Neem/Tulsi extract + Maize starch - Compatible and useful for wet granulation.

Neem/Tulsi extract + SSG - Compatible; supports rapid disintegration.

Neem/Tulsi extract + Magnesium stearate - Compatible in small amount.

Neem/Tulsi extract + Talc - Compatible and improves granule flow.

6] Prepartion of Granules via Wet Granulation Technique

Procedure :Sieve neem extract, tulsi extract, lactose, DCP, and SSG separately. Mix neem extract + tulsi extract + lactose + DCP + SSG uniformly. Prepare 10% maize-starch paste using maize starch and purified water.Add starch paste slowly to the powder blend and mix until a cohesive wet mass forms.Pass wet mass through sieve No. 12 or 16 to make wet granules. Dry granules at 40–50°C until they are dry.Pass dried granules through sieve No. 20. Add talc and magnesium stearate; mix gently for 2–3 minutes.Compress the lubricated granules into 420 mg tablets.

Preparation of 10% starch paste

Procedure :Take maize starch as per needed.Mix it with cold water to make a smooth slurry.Heat few mL dis.water separately. Add starch slurry slowly into hot water while stirring.Heat and stir for 3-5 minutes until a smooth paste forms , Cool slightly.Add the paste slowly to the powder mixture to preapare wet granules.

Evalution of Granules

Preformulation studies

Bulk density : Bulk density was carried out in 100 ml dried measuring cylinder. Pouring of dried granules in measuring cylinder and calculated by using the following formula;

Bulk density = Mass of the granules/Bulk volume of the granules

Tapped density ; Tapped density was carried out by pouring of dried granules in 100 ml measuring cylinder.100 tapping was done, note down the volume and calculate by using the following formula;

Tapped density= Granules weight/Volume of tapped granules

Hausner’s ratio : Hausner’s ratio is the ratio of the tapped density of granules to the bulk density of granules. Calculated by using the following formula.

Hausner’s ratio= Tapped density/Bulk density

Carr’s index

Carr’s index or compressibility index is determined by the following formula

Carr’s index[%]= Tapped density –Bulk density /Tapped density ×100

Angle of repose : Angle of repose was determined by using the funnel method. Following formula was used to calculate the angle of repose

ϴ = Tan-1[h/r]

Where  h = height of granule cone formed. , r = radius of the granule cone formed.  

Table - 2: Scale of flowability

 7] Compression of Tablets : Check granules ensure that are dry and free –flowing. Clean the machine and Select punch and die . 8-10mm for 420mg tablet.Fill the hopper.Adjust tablet weight.Set Compression force then Prepare trial tablets.Check trial tablets ,compress full batch.Collect and store tablets .

Physical evalution of tablets ;

General appearance : The general appearance and color of tablets were found by visual  determination.

Weight variation test : The weight variation test was performed by following procedure. Weigh 20 tablets individually and consider as X1, X2,X3,…. .X20.  Determine the average weight of 20 tablets X= (X1+X2+X3+….+X20)/20. The individual weight was compared with the upper limit and lower limit. Not more than two of the tablets differs from the average weight by more than the % error listed, and no tablets differ by more than double that percentage. Results are shown in table 8. 

Table - 3 : Weight variation tolerance

Hardness and thickness test: For each formulation, the hardness and thickness of 20 tablets were determined. Hardness test was determined by Monsanto hardness tester and the thickness of tablets was determined by Vernier Calipers. Results are shows in table 8.

Friability test : Friability of a tablets can determine in a laboratory by Roche friabilator. The friabilator consists of plastic chamber that rotates at 25rpm, dropping the tablets through a distance of six inches in the friabilator, which is then operated for 100 revolutions. The tablets are reweighed. Compress tablets loss less than 0.5% to 1.0% of the tablet weight are considered acceptable. Results are shown in table 8.

Disintegration time : This test was a time required for the tablet to separate into particles, the disintegration test measure only of the time required under a given set of a conditions for a group of tablets to disintegrate into particles. This test was performed to identify the disintegration of tablet in a specific time period. Results are shown  in table 8.

Tablets - 4 : Formulation chart for the preparation of table

RESULT AND DISCUSSION

5.1 Physicochemical Evalution of plant materials

5.1.1 Percentage yield of all the hydroalvoholic plant extract Roche Friabilator

The percentage yield of HA plant extract are given in the Table.

• Dried powder used     = 250g
• Dried extract obtain    = 20g
• %yield = 20/250×100 = 8%         

Table - 5 : Extract obtain

5.1.2. Preliminary Phytochemical screening of HA plant extract

Table - 6: Phytochemical Test

5.1.3 . Evaluation of Powder Blend/ Granules /Preformulation Parame

Table -7:  Preformulation Evalution

    5.1.4. Post Formulation Parameters

Table - 8:  Post formulation Evalution

5.1.5. Experimental UV- Visible absorption Data for Neem extract

Table - 9:  UV-absorption /Wavelength for neem extract

Figure- 5.1: UV-Graph of Neem extract

Table - 10 : UV- absorption / Wavelength for Tulsi

Figure- 5.2: UV- Graph of  Tulsi extract

5.1.6 Determination of λmax.

The absorotion spectrum of pure drug was scanned 200-800 nm with 10mg/ml prepared in ethanol .The λmax. of  Neem and Tulsi was to be 359 and 412 nm.

5.1.7 Observation table :     

Table- 11: Compound identified in Neem extract

                              Table- 12 : Compound identified in Tulsi extract