Thuja occidentalis (commonly Morpankhi, Cupressaceae) is a dioeciously evergreen tree or shrub that has been employed for a number of antipyretic, antitussive, astringent, diuretic, cooling, and stomach functions. The phytochemicals labdane and isopimaran, as well as monoterpenoids and sesquiterpenoids found in the essential oils of various plant components, were isolated from various portions of Platycladus orientalis.  Two monolognol derivatives derived from pollen and two diterpenoids from pericarp and leaves. The most advantageous elements of T. orientalis oil are carnphor, fenchone, isothujone, and thujone. Treatments using a variety of complementary therapies are gaining popularity. The use of plants to treat a variety of illnesses is important in this setting. Despite being widely utilised around the world, several governments do not formally recognise folk treatments. More than 80% of people in Asia and Africa  utilize  plant extracts to treat a range of diseases.

Figure:-1 Plant of Thuja occidentalis

Tree:-

The tree is modest and slow-growing, reaching a height of 15-20 m (49-66 ft) with a trunk diameter of 0.5 m (1 ft 8 in). On rare occasions, older trees can reach heights of 30 m (98 ft) and diameters of 2 m ( 6 ft 7 in).An evergreen tree is Thuja orientalis. Hardy Thuja orientalis is a small to medium-sized tree that typically does not exceed a height of 20 meters.

Seed cones:-

The narrow, yellow-green, developing to brown seed cones measure 9–14 mm in length, 4-5 mm in width, and have six to eight overlapping scales. About eight seeds are included in each. The male cones are tiny, barely noticeable, and found at the ends of twigs. When mature, female cones have lengths of about 1 to 2 cm and open in the first year, at 6 to 8 months of age.

Leaves:-

The leaves of Hardy Thuja orientalis are predominantly scale-like, ranging in length from 1 to 10 mm, except for the tiny seedlings in their first year, which have needle-like leaves. These scale leaves are arranged in four rows along the branches, alternating in decussate pairs. The leaves have a rough texture, with a lighter color on the lower surface and a darker color on the upper surface. During winter, the leaves may acquire a coppery tint. They are arranged in flattened, fan-shaped clusters in four rows, and resin glands are grouped oppositely within these clusters.

Bark:-

Reddish-brown in colour, it has ridges and peels off in long, slender strips. Evergreen thuja trees range in height from 10 to 200 feet (3 to 61 metres) and have reddish-brown, fibrous-textured bark. The shoots of Hardy Thuja orientalis are flat and typically have only one plane of side shoots. This means that the side shoots emerge from the main shoot in a single plane, rather than being arranged in multiple planes or directions. This gives the shoots a relatively flat appearance.

Roots:-

Small trees' roots can reach a depth of up to 8 inches, whereas the roots of larger trees can extend as far as 18 to 24 inches. They need direct sunlight, but they can also grow in dark environments.

General uses of the plant:-

• It is traditionally used to cure coughs.
• Like many other members of the Cupressaceae family, Thuja can cause allergic reactions that include breathing issues, skin and eye.
• The plant has been extensively studied and shown to possess various beneficial properties, such as anti-epileptic, antiviral, antiallergic, antibacterial, antioxidant, and hair-growth-promoting effects.
• Scalds and burns can be treated with the root and bark.
• The leaves are used to treat a variety of ailments, including those that are antibacterial, astringent, antipyretic, stomachic, antitussive, antipyretic and diuretic.
• The usage of trees in general for various medicinal products and aromatherapy concoctions dates back a long way
• It has been used externally to cure fungal skin illnesses including ringworm and thrush, reduce the discomfort associated with arthritis and rheumatism, and eradicate anal or vaginal warts.
• The tree is known as arbour vitae or the "tree of life" because of its vitamin C-rich leaves and twigs, which are beneficial in preventing or treating scurvy.

Biological activities

Anti-Inflammatory Activity

Inflammation is an essential defence mechanism that occurs in response to tissue damage, aimed at facilitating tissue repair. The activation of macrophages is initiated through neutrophil extravasation within the capillary network, leading to the production of various proinflammatory cytokines such as TNF-?, interleukins, and interferons, which play crucial roles in regulating inflammatory responses. Although limited, there have been some studies investigating the anti-inflammatory properties of components found in T. occidentalis.

Antifungal Activity

In a recent study, the essential oil from Thuja occidentalis leaves and cones showed antibacterial efficacy against yeast like Candida albicans, fungus like Aspergillus flavus and Aspergillus niger, and Gram-positive bacteria like Staphylococcus aureus and Listeria monocytogenes. Gram-negative microorganisms including Aeromonas hydrophila, Salmonella typhimurium, Escherichia coli, and Pseudomonas. Antifungal properties may be found in essential oils.

Antiviral Activity

It has been demonstrated that T. occidentalis polysaccharides contain antiviral and immune-stimulating qualities, with the capacity to lessen HIV-1 and influenza. A. At a concentration of 625 g/mL, thuja polysaccharides (TPS) suppressed HIV. It was demonstrated that freshly infected MT-2 cells had no detrimental effects on MT-4 cells and displayed lower expression of the HIV-1 specific antigen. emphasised the effect of T. occidentalis' high-molecular-weight polysaccharide on HIV-1. An in vitro study found that an isolated T. occidentalis component increased the number of cells making antibodies.

Hair growth promoting activity

In East Asia, T. orientalis has long been used to treat people who have baldness and hair loss.  According to Zhang et al.'s research, T. orientalis hot water extract stimulated the anagen phase in telogenic C57BL/6 N mice, promoting hair growth.  In contrast to either the control group or the group given 1% minoxidil, topical treatment of T. orientalis extract generated an earlier anagen phase and prolonged the mature anagen phase.

Antimicrobial activity:

The essential oil from the seed coats of T. orientalis against six bacterial pathogens, including Aspergillus niger, Aspergillus fumigatus, Rhizopus oryzae, Fusarium psidi, and Curvularia lunate, as well as five fungal pathogens.  The oil had good to moderate action against each of the six test microorganisms. The monoterpene ketones a- and ß-thujone, fenchone and sabinene, as well as the diterpenes beyerene and rimuene, were the predominant components in all samples. S. aureus, S. epidermidis, P. aeruginosa, E. cloacae, K. pneumoniae, E. coli, Candida albicans, C. tropicalis, and C. glabrata were tested for essential oil's antimicrobial activity.Thuja leaf essential oil's antibacterial properties were investigated by Malik and Singh 28 against microorganisms isolated from urinary tract infections. While E. coli was discovered to be a resistant bacteria, P. mirabilus and S. aureus were determined to be the most susceptible bacteria. 

Antibacterial activity:

Antimicrobial substances can be found in essential oils, which are helpful sources.  It is commonly known that plants contain antibacterial elements since these elements have served as a source of inspiration for new medication compounds used in herbal remedies for human health.  T. orientalis contains large amounts of alpha, beta, and gamma thujaplicin, which may have the ability to chelate Solmonella typhimurium at low doses.Streptococcus mutans was resistant to the antibacterial effects of T. orientalis, according to Chen and colleagues. The antibacterial and antifungal effects of Vernonia anthelmintica, Dryopteris chrysocoma, and Trachyspermum ammi were tested in vitro on Pseudomonas aeruginosa, Yersinia aldovae, Citrobacter, Shigella flexneri, E. coli, and Staphylococcus aureus in the alcohol concentrate made from Thuja occidentalis twigs. Antibacterial testing was done on six microscopic species, including Citrobacter, Escherichia coli, Staphylococcus aureus, Yersinia aldovae, Shigella flexenari, and Pseudomonas aeruginosa all shown strong movement against all bacteria. These concentrations were tested for their antifungal efficacy against six growths: Candida albicans, Saccharomyces cereviciae, Aspergillus parasiticus, Trichophyton rubrum, Macrophomina, and Fusarium solani. When used against several parasite strains, the concentrates demonstrated important results.

MATERIALS &  METHOD

Cultivation Collection

Thuja is cultivated outdoors in a cool, humid microclimate in hedges, open ground, pots, solitary and group plantings, and forest protection strips. In November or March, these plants are set in place permanently. They favour soil that is deep, slightly damp, and well-drained. Thuja is cultivated for hedges with a 60–70 cm spacing between plants. Use a peat substrate, fertile ground, and organic fertilisers in the amount of 30–50 g per bucket of soil for growing it in pots or flower beds. Practise liquid top dressing with mineral fertilisers beginning in the second year of life. Thuja occidentalis fresh roots were gathered from locations in and around Lucknow. The gathered roots were cleaned and then chopped into little pieces so they could be turned into powder. To make powder, little bits of root were smashed in a mortar and pestle. Thuja occidentalis root powder was sieved through a 40-no sieve to obtain a fine powdered medication.

Microscopic of Thuja occidentalis

• Outermost layer of the circular roots is thick walled epiblema with many root hairs.
• Epiblema is followed by many layers of parenchymatous cortex.
• Inner to the cortex is present a layer of endodermis and many layers of pericycle.
• Vascular bundles are radially arranged and diarch to tetrarch with exarch protoxylem.
• Protoxylem is bifurcated (Y- shaped) towards the periphery, and in between each bifurcation is present a resin cannel.
• Pith is poorly-developed or absent.

 Drying and Powdering of Thuja occidentalis root

After cutting the roots into small pieces, it was washed, then the roots were dried at 100 0C temperature for two weeks, and then fine particles help with a motor pestle, it was converted into fine powder, then that powder was grinded in a mixer and that grinder powder was passed through a 40 no Sieve filter it and collect the powdered drug and the drug is ready.

Selection of solvents

Solvents are playing the major role in extraction process. There are two type solvent used in the extraction process non-aqueous solution not present in water ( ethyl acetate,ecetone, ethanol)  and Aqueous solution it means present in water ( methanol, water).  Based on the active constituents of Thuja occidentalis, solvents like -

• Ethyl acetate
• Acetone
• Ethanol
• Methanol
• Water

Extraction by Soxylation

On a small scale, the soxhlet device is utilised for the extraction.  A Soxhlet extractor, a flask and a reflux condenser make up this apparatus.

       
           
       
    Figure 6:- Soxhlet apparatus

The thick tube of the extractor was filled with the coarsely ground Thuja occidentalis powder that was contained in the filter paper thimble.   In the extraction process for the plant medicine, two solvents—a non-aqueous solution and an aqueous solution—were used: ethyl acetate, acetone, ethanol (F1), and methanol, water (F2).  During this time, soluble constituents were removed as the solvent liquid level slowly rose. The liquid is syphoned into the flask when its level reaches the syphon's top.  Repeating the previous procedure, a portion of the solvent from the solution was once more evaporated, leaving the contents in the flask itself. Additionally, an ethyl acetate and methanol extract was used to remove the entire plant's contents.  The plant was then taken out of the thimble and dried outside. After the plant is prepared to be treated with ethanol to remove the active components from the plant, pet ether from the plant is removed.    The flask containing the solvent ethanol was filled and heated. Up until the last solute was extracted, the same procedure was repeated repeatedly. To get rid of the solvent ethanol, the extractive liquid that included active ingredients was treated with a rotary film evaporator.

       
           
       
    Figure 7:- Extraction process

       
           
       
    Figure 8:- Extraction of plant drug F1 & F2

Phytochemical Analysis

Thuja occidentalis is analysed using phytochemistry to identify phytochemicals like alkaloids, gycosides, carbohydrates, saponin, flavonoids, and fixed oil.

Alkaloids

The extracts sample was treated with potassium bismuth iodide solution (Dragrndroffs reagents). Formation of orange red precipitate indicated the presence of alkaloids.

Carbohydrates

Dissolved 2gm extracts in 5ml water and filter it with filter paper. The filtrate was then mixed with concentration sulphuric acid and adds few drops of Molish’s reagent. Indication of pink or violet colour shows the availability of carbohydrates.

Phenolic compound

Take 2ml extract dissolved in H2O and add 3-4 drops of ferric chloride solution and indicated the bluish black colour shows availability of phenols.

Tannins test

Take 0.5gm of dried powdered and boiled 0.5gm sample in 20ml of water in a test tube and filter the above mixture. Add few drop of 0.1?rric chloride and developed brownish green or blue black colour shows availability of tannins.

Flavonoids

Treat the extract with few drops of lead acetate solution. Flavonoids were present because yellow precipitate formed.

Glycosides

In the Keller- Killani test, 2 ml of plant extract solution was treated with 0.5 ml of glacial acetic acid solution containing 2-3 drops of ferric chloride. One millilitre of concentrated sulphuric acid was added to the test tube, and the presence of deep blue colour indicated the presence of cardiac glycosides.

Fixed oils and fats

One drop of concentrated sample was placed between two filter sheets and kept undisturbed for 2 minutes. Development of oil stains on paper shows presence of fat and oil

Saponins

2ml of the sample extract was mixed in 20ml of water and shake it in a graduated cylinder for 15 min. Emergence of 1 cm layer of foam shows the availability of saponin.

       
           
       
    Figure 9: – Phytochemical test (Alkaloids, Glycosides, carbohydrates, phenolic compound, tannins, saponin

ANTIBACTERIAL TEST PREPARATION

Antibacterial-Zone Inhibition Test- B. Subtilis

Disc Diffusion Method                                                                           

Sample – F1
       
           
       
    

Amount present per disc in µg

Dispensed Volume- 10µL

Positive Control- 10 µg

Figure:-10 Disc Diffusion Plate- Sample – F1

Sample – F2

Amount present per disc in µg

Dispensed Volume- 10µL

Positive Control- 10 µg

Figure:-11 Disc Diffusion Plate- Sample – F2

Antibacterial -Zone Inhibition Test- E.coli

Disk Diffusion Method                                                       

Sample- F1                                                                                              

Amount present  per Disk in µg

Dispensed Volume- 10µL

Positive Control -  10µg

Figure:- 12 Disc Diffusion Plate- Sample- F1

Sample- F2

Amount present  per Disk in µg

Dispensed Volume- 10µL

Positive Control -  10µg

Figure:- 13  Disc Diffusion Plate- Sample- F2

Antibacterial-Zone Inhibition Test- S.aureus

Disk Diffusion Method

Amount present  per Disc in µg

Dispensed Volume- 10µL

Positive Control -  10µg

Amount present per Disc in µg

Dispensed Volume- 10µL

Positive Control - 10µg

Figure:-15 Disc Diffusion Plate- Sample- F2

Antibacterial-Zone Inhibition Test- P.aeruginosa

Disc Diffusion Method

Sample- F1

Dispensed Volume- 10µL

Positive Control - 10µg

Figure: - 16 Disc Diffusion Plate- Sample- F1

Disc Diffusion Method

Sample- F2

Amount present  per Disc  in µg

Dispensed Volume- 10µL

Positive Control - 10µg

Figure:-17 Disc Diffusion Plate- Sample- F2

Final extract of Thuja occidentalis

The root extract of thuja occidentalis was successfully accomplished by using Ethyl acetate, acetone, ethanol (F1) and methanol & water (F2) was using soxhlet extraction at 120 ºC for 48 hrs. The detail of extraction process was described in above chapter. The prepared extracts were evaluated for several phytochemical characteristics, including Carbohydrate, Tannins, Phenolic compound, Fixed oil & fats, Glycosides, Saponin and the result of these tests showed in table no…and The root extract were further evaluated against various bacterial species such as E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, and B. Subtilis. The  result of anti-bacterial activity was discussed in bellowing.

Figure:-18

Phytochemical analysis of herbal extract:-

Table:-2 Phytochemical analysis

Antibacterial activity – B. Subtilis F1

Table:-3 Anti- Bacterial Activity- B. Subtilis F1

Antibacterial bioassy – B. Subtilis F2

Table:-9 Anti- Bacterial Bioassay– P.aeruginosa F1

Graph:-7 Anti- Bacterial Bioassay– P.aeruginosa F1

Antibacterial bioassay – P. Aeruginosa F2

In this work has attempted to make herbal extract (F1 & F2) using Thuja occidentalis roots.  The Non-aqueous soxhlet extract (F1) indicated the highest percentage yield (80%) and the aqueous soxhlet extract (F2) indicated the low percentage yield (76%) compare to F1. The root extract were evaluated against various bacterial species such as E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, and B. Subtilis and the Result concluded that the all extract solution exhibited antibacterial effect and exhibited maximum activity with zones of inhibition from on E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, and B. Subtilis. respectively. The generated extracts were further evaluated for several phytochemical characteristics, including Carbohydrate, Tannins, Phenolic compound, Fixed oil & fats, Glycosides, Saponin and the result of these tests showed in table no.  According to literature survey the roots of Thuja occidentalis possess the necessary medicinal characteristics and they utilized ethno-botanically and offer a lot of potential for creating antibiotics for bacterial illnesses. The herbal extract was developed by using an efficient, simple soxhlet extraction method with highest percentage yield ranging from 70-80% discussed in above paragraph.  Literatures has already shown that plant extracts of Thuja occidentalis are not toxic to humans, Further study should focus on its use to treat bacterial infection in low- and middle-income nations, as well as the extraction method used for that purpose. Using the same conditions for all approaches and it is crucial to conduct experimental tests to confirm the efficacy of the thuja occidentalis. Thuja roots have been used to cure a variety of illnesses, the therapeutic uses of this plant may be connected to the discovery of bioactive substances. Thus, the result also concluded that the prepared extract has potential source for treating bacterial infections may be investigated that is:-