Antimicrobial Activity and Isolation of Lawsone from Lawsonia inermis using Column Chromatography

 

Utkarsha Shivsharan*, Yash Kothari

DSTSM’s College of Pharmacy, Vijapur Road-1, Jule Solapur, Solapur. Maharashtra, India. 413003.

 

ABSTRACT:

Henna is most commonly used for coloring the hair from more than 5000 years. It contains Lawsone which interacts with keratin present in skin and hair by Michael addition which results into permanent stain. Chemically Lawsone is 2-hydroxy-1,4-naphthoquinone found in the leaves of the henna plant as well as in the flower of water hyacinth. But the Lawsone isolation from Lawsonia inermis can be difficult due to its easily biodegradable nature. Hence isolation of Lawsone is done by using Column chromatography. The isolated extract was also tested for antimicrobial activity keeping Vancomycin as a standard. It has shown prominent Antimicrobial activity.

 

 

 

INTRODUCTION:

Humans have used henna extracts containing lawsone as hair and skin dyes for more than 5000 years. Lawsone reacts chemically with the protein known as keratin in skin and hair, in a process known as Michael addition, resulting in a strong permanent stain that lasts until the skin or hair is shed. The darker colored ink is due to more lawsone-keratin interactions occurring, which evidently break down as the concentration of lawsone decreases and the tattoo fades. Lawsone strongly absorbs UV light, and aqueous extracts can be effective sunless tanning and sunscreens. Chemically, lawsone is similar to juglone, which is found in walnuts.

 

Lawsone (2-hydroxy-1,4-naphthoquinone), also known as hennotannic acid, is a red-orange dye present in the leaves of the henna plant (Lawsonia inermis) as well as in the flower of water hyacinth (Eichhornia crassipes).

 

Lawsone isolation from Lawsonia inermis can be difficult due to its easily biodegradable nature.

 

MATERIALS AND METHODS:

Lawsone was procured from the leaves of Henna from the local garden located in the premises of the Dayanand College of Arts and Science. Methanol and Chloroform were used as solvents in the column chromatography for running process were obtained from Vijaya Chemicals, Solapur.

 

Isolation of Lawsone:

1.     Preparation of Alcoholic Crude Extract:

The leaves of the shed dried henna were powdered well in a mortar. Further this powered Crude drug was added with methanol and incubated at 37ºC for 5 days. During this incubation period, the polar as well as non-polar compounds which are to be isolated from Lawsonia inermis get separated in the black supernatant in the methanolic crude extract. Once the incubation period was lapsed, the methanolic crude supernatant was separated from the sediment and was allowed to air dry without sunlight to interfere for at least 2 days. Silica gel for Column Chromatography (60-120 Meshed) was used with ensured continuous mixing in order to make a cake like consistency of the silica. Once this was prepared, the crude silica alcoholic extract was allowed to be introduced into the column in which chromatography was to be performed and solvents were allowed to run throughout. With this, the filling process of the column was completed.

 

2.     Selection of Solvents:

Solvent systems for use as mobile phases in CC can be determined from TLC experiments. Normally, a separation will begin by using non-polar or low polarity solvent, allowing the compounds to adsorb to the stationary phase, then slowly switching the polarity of the solvent to desorbs the compounds and allow them to travel with the mobile phase. The polarity of the solvents should be changed gradually.

 

Some typical solvent combinations are ligroin-dichloromethane, hexane-ethyl acetate and hexane-toluene. Often an experimentally determined ratio of these solvents can sufficiently separate most compounds. Solvents such as methanol and water are normally not used because they can destroy the integrity of the stationary phase by dissolving some of the silica gel.

 

3.     Packing of the column:

There are several acceptable methods for packing a column. These include dry packing (there are two versions of dry packing discussed here) and the slurry method. Dry packing is the method of choice for a microscale column beginning by filling the column with a nonpolar solvent. Slowly add the powdered alumina or silica while gently tapping the side of the column with a pencil. The solid should “float” to the bottom of the column. For the second dry pack method, the stationary phase is deposited in the column before the solvent. In this case filling the column to the intended height with the stationary phase is done and then slowly addition of the nonpolar solvent. The solvent should be added slowly as to avoid uneven channeling. This method is typically used with alumina only, since silica gel expands and does not pack well with this dry method. The slurry method is often used for macroscale separations. Combine the solid stationary phase with a small amount of nonpolar solvent in a beaker. Thoroughly mix the two until a consistent paste is formed, but is still capable of flowing. Pour this homogeneous mixture into the column as carefully as possible using a spatula to scrape out the solid as you pour the liquid. The slurry method normally gives the best column packing, but is also a more difficult technique to master. Whether the dry or slurry method is chosen, the most important aspect of packing the column is creating an evenly distributed and packed stationary phase. As mentioned, cracks, air bubbles and channeling will lead to a poor separation.

Once the column is loaded, open the stopcock and allow the solvent level to drop to the Top of the packing, but do not allow the solvent layer to go below this point. Allowing this solvent level to go below the stationary phase, (known as letting the column to “run dry,”) should always be avoided. Since it allows air bubbles and channel formation to occur leading to a poor separation.

 

4.     Adding the solvent:

Once the packing is complete, the sample can be loaded directly to the top of the column. Normally, a minimum amount of a polar solvent, 5-10 drops, is used to dissolve the mixture. The solution is then carefully added to the top of the column using a pipette without disrupting the flat top surface of the column. A thin horizontal band of sample is best for an optimal separation.

 

After the sample is loaded, a small layer of white sand is added to the top of the column. This will help to keep the top of the column level when adding solvent eluent. Once the mixture is added and the protective layer of sand is in place, continuously add the solvent eluent while collecting small fractions at the bottom of the column. Using a pipette to add the first bit of solvent on top of the packing, sample, and sand will minimize disturbance of the column and diluting the sample.

 

Collecting small fractions (1-3 mL) is important to the success of your column separation. Fractions that are too small can always be pooled together; however, if the collected fractions are too large, you may get more than one compound in any particular fraction. If this occurs, the only way to complete the separation is to redo the chromatography. Since column chromatography is time consuming, collecting large fractions is discouraged.

 

Monitoring The column:

If the mixture to be separated contains colored compounds, then monitoring the column is very simple. The colored bands will move down the column along with the solvent and as they approach the end of the column, collect the colors in individual containers. Use the color as your guide. However, most organic molecules are colorless. In this case, the reaction must be monitored by TLC. Spot each fraction on a TLC plate. Four or five fractions can be spotted on a single TLC plate. Develop the plate and use the observed spot or spots to determine which compound is in each of the collected fractions. Spotting some of the starting material or the product (if available) on the TLC plate as a standard will help in the identification.

 

Isolating the Separated Compounds:

Once you believe all the materials have been removed from the column, the colors of the materials or TLC results should indicate which fractions contain the compound(s) you are interested in isolating. Combine the like or same fractions and evaporate the solvent. The pure separated compound will be left behind. Recrystallization may be used to further purify a solid product. However, on a milligram scale, there is usually not enough.

 

Pharmacognosy of Henna:

Synonym: Mehendi, Egyptian privet.

Biological Source: It Consist of Shed dried leaves of the plant Lawsonia inermis belonging to the family Lyrthaceae. Henna is a medium sized herb consisting of many branches and small white fragrant flowers. The leaves of henna are greenish brown in colour with a characteristic odour and have a bitterish astringent taste. Henna can be used in the form of powder, decoction and pastes.

 

Geographical Source:

Henna is popular by the name “mehendi” and is grown throughout India. Henna is also widely popular in countries like Africa, Egypt, Sudan, Caribbean Islands, South Florida And China.

 

Morphological Characteristics:

Lawsonia inermis is a glabrous branched shrub or small tree (2 to 6 m in height). Leaves are small, opposite, entire margin elliptical to broadly lanceolate, sub-sessile, about 1.5 to 5 cm long, 0.5 to 2 cm wide, greenish brown to dull green, petiole short and glabrous acute or obtuse apex with tapering base. New branches are green in colour and quadrangular, turn red with age. Young barks are greyish brown, older plants have spine-tipped branchlets (Fig. 1). Inflorescence has large pyramid shaped cyme. Flowers are small, numerous, aromatic, white or red coloured with four crumbled petals. Calyx has 0.2cm tube and 0.3 cm spread lobes. The fruits are small, brown globose capsule, opening irregularly and split into four sections with a permanent style.

 

Ethnopharmacology:

Lawsonia inermis is a well known ethnomedicinal plant used cosmetically and medicinally for over 9,000 years. Its use in the Indian traditional folk medicines is well documented. Table 1 indicates the use of different parts of L. inermis in traditional system of medicines.

 

Plant Parts	Traditional Uses (as/in)
Root	Bitter, depurative, diuretic, emmenagogue, abortifacient, burning sensation, leprosy, skin diseases, amenorrhoea, dysmenorrhoea and premature greying of hair.
Leaves	Bitter, astringent, acrid, diuretic, emetic, edema, expectorant, anodyne, anti-inflammatory, constipating, depurative, liver tonic, haematinic, styptic, febrifuge, trichogenous, wound, ulcers, strangury, cough, bronchitis, burning sensation, cephalalgia, hemicranias, lumbago, rheumatalgia, inflammations, diarrhoea, dysentery, leprosy, leucoderma, scabies, boils, hepatopathy, splenopathy, anemia, hemorrhages, hemoptysis, fever, ophthalmia, amenorrhoea, falling of hair, greyness of hair, jaundice.
Flowers	Cardiotonic, refrigerant, soporific, febrifuge, tonic, cephalalgia, burning sensation, cardiopathy, amentia, insomnia, fever.
Seeds	Antipyretic, intellect promoting, constipating, intermittent fevers, insanity, amentia, diarrhoea, dysentery and gastropathy.

 

Phytochemistry of Lawsonia inermis:

Much work is done in the field of phytochemical investigation of the plant. The chemical constituents isolated from L. inermis are napthoquinone derivatives, phenolic compounds, terpenoids, sterols, aliphatic derivatives, xanthones, coumarin, fatty acids, amino acids and other constituents. Phytochemicals reported in L. inermis L. are listed in (Table 2)

 

Compounds	Plant Parts
Napthoquinone derivatives Lawsone (2-hydroxy 1,4-naphthoquinone)	Leaves
1,3-dihydroxy naphthalene, 1,4-napthaquinone, 1,2-dihydroxy-4-glucosylnaphthalene	Leaves
Isoplumbagin	Stem bark
Phenolic conpounds Lawsoniaside (1,3,4-trihydroxynaphthalene 1,4-di-β-D-gluco-pyronoside), Lalioside (2,3,4,6-tetrahydroxyacetoxy-2-β-D-glucopyranoside) Lawsoniaside B (3-(4-O-a-D-glucopyranosyl-3,5-dimethoxy) phenyl-2E-propenol), syringinoside, daphneside, daphnorin, agrimonolide 6-O-β-D-glucopyranoside, (+)-syringaresinol O-β-D-glucopyranoside, (+)-pinoresinol di-O-β-D-glucopyranoside, syringaresinol di-O-β-D-glucopyranoside, isoscutellarin	Bark, Leaves
Terpenoids 3β, 30-dihydroxylup-20(29)-ene (hennadiol), (20S)-3β, 30-dihydroxylupane, Lupeol, 30-nor-lupan-3β-ol-20-one, betulin, betulinic acid, lawnermis acid (3β-28β-hydroxy-urs-12,20-diene-28-oic acid) and its methyl ester	Bark, Seeds
Sterols Lawsaritol (24β-ethycholest-4-en-3β-ol) Stigmasterol and β-sitosterol	Roots, Leaves
Aliphatic constituents 3-methyl-nonacosan-1-ol, n-tricontyl n-tridecanoate	Stem bark
Compounds	Plant Parts
Xanthones Laxanthone I (1,3 dihydroxy-6,7 dimethoxy xanthone), Laxanthone II (1-hydroxy-3,6 diacetoxy-7-methoxyxanthone), Laxanthone III (1-hydroxy-6-acetoxy xanthone)	Whole plant
Coumarins Lacoumarin (5-allyoxy-7-hydroxycoumarin)	Whole plant
Flavonoids Apigenin-7-glucoside, apigenin-4-glycoside, luteolin-7-glucoside, luteolin-3-glucoside	Leaves
Essential oil -(Z)-2-hexenol, linalool, α ionone, β ionone, α-terpineol, terpinolene, δ-3-carene and γ-terpineol	Leaves
Other chemical constituents Hennotannic acid, glucose, gallic acid, amino acid Trace metal - Cu, Ni, Mo, V, Mn, Sr, Ba, Fe and Al Minerals - Na2O, CaO and K2O	Whole plant

 

Pharmacological Actions of L. inermis:

Several researchers have reported the different biological actions of L. inermis in various in-vitro and in-vivo test models. Henna leaves, flower, seeds, stem bark, roots have been found to exhibit antioxidant, antidiabetic, hepatoprotective, hypoglycemic, antimicrobial, anticancer and wound healing properties. These are described in greater details in the following sections.

 

Chemistry of Lawsone:

2-Dimensional structure:

 

Chemical Names:

·       2-hydroxy-1,4-naphthoquinone

·       2-hydroxynaphthoquinone

Molecular Formula: C₁₀H₆O₃

Molecular Weight: 174.155g/mol

 

RESULT AND DISCUSSION:

Quantitative Estimation of Lawsone in Fresh and dried leaves of Lawsonia inermis:

Quantitative estimation of Lawsone in fresh and dried leaves was carried out using spectrophotometric method in which 100mg leaves of L. inermis were soaked in methanol for atleast 2hours. Thereafter, the mixture was centrifuged at 5000rpm for 20 minutes and the clear supernatant was separated into another test tube. The absorbance of supernatant was read out at 452 nm. A calibration curve was prepared by plotting the concentration versus absorbance at the same wavelength in the concentration range 10-200µg/ml of pure lawsone. The lawsone content in fresh and dried leaves was then calculated in µg/ml. All the observations were taken into triplicate.

 

Values: 5.3µg/ml-6.9µg/ml.

 

RESULT:

The antimicrobial effect observed for this class of naphthoquinones i.e. Lawsone against Staphylococcus aureus and E. coli is presented in the table below. The effect of this compound on MRSA i.e. Methicillin resistant Staphylococcus aureus were also inhibited by Lawsone where as they showed resistance to the standard marketed drug Vancomycin. The compound also inhibited the growth of certain gram-negative strains like Salmonella typhi also. The presence of 1% serum albumin in the culture media often caused increase in MIC values although the compound was potent enough to exhibit it antimicrobial activity against these strains.

 

Strains used	Concentration (µg)	Zone of inhibition (mm)
MRSA S. aureus	20	23.8
E. coli	25	29.7
S. typhi	20	26.4
S. typhi(vancomycin)	75	22.6

 

CONCLUSION:

From the results obtained from the above data, it can be concluded that the antimicrobial activity was been possessed by the Lawsone. The results collected showed greater and much more zones of inhibition than those of that showed by the standard marketed drug antibiotic Vancomycin. The zones of inhibition shown by vancomycin against Salmonella typhi ranged to across 22.6 mm and that showed by the lawsone was 26.4 mm. Hence, lawsone can be used and introduced in the market since it possesses quite good activity enough to kill micro-organisms that may cause troublesome problems to humans. However, the quantity of lawsone used in this experiment is much more less and is quite economical than production of drugs like Clotrimazole, Vancomycin, Oxiconazole, Miconazole since these drugs require special manufacturing techniques. Isolation of 5-6µg of lawsone hardly requires the time of maximum 42 hours and is natural in origin. Hence there will not be any admixture of other compounds rather than synthetically prepared drugs.

 

 

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Received on 08.07.2020         Modified on 27.07.2020

Accepted on 14.08.2020  ©AandV Publications All right reserved