INTRODUCTION:

The lotus, commonly known as kamal or Padma, is an aquatic perennial plant that solely grows in the water for more than two growing seasons. Worldwide, there are only two species of Nelumbo lutea Wild and N. nucifera. This N. nucifera, the Indian lotus, is found throughout Asia and Australia, whereas N. lutea, considered to be a subspecies of N. nucifera, the American lotus or water chinquapin, occurs in eastern and southern North America.

It is generally found in warm temperate to tropical climate. In other words, it is found in India (central and northern), China and Vietnam to Japan, Malaysia, New Guinea and Australia^1,2. The lotus is considered to be given a sacred status amongst the Indian culture. Its leaf is grown at the bottom of ponds but emerges above the water surface, untouched by the contaminants of the dirty water on which it grows^3–5.

The hydrophobic and water repellent properties of many plant leaves have been known for a long time. With the help of scanning electron microscope (SEM), studies were carried out in the past 30 years that revealed the leaf surface hydrophobicity which is related to the microstructure⁶. Actually all primary parts of plants are covered by a cuticle composed of lipids embedded in a polyester matrix which makes the cuticle hydrophobic in most cases. The outer single layer of cell group covering a plant is known as epidermis⁴. The protective waxy covering produced by these cells is known as cuticle, which is composed of an insoluble cuticular membrane covered with epi-cuticular wax, which are mixture of hydrophobic aliphatic components, hydrocarbons with chain length typically in range C16 to C36 such as paraffin^7-9.

To determine what extent a given surface is hydrophobic, the shape of water droplet i.e. the contact angle of the water droplet to the surface is measured. The more hydrophobic a surface, the more will be the contact angle and the more spherical will be the water droplet. The lotus plant’s leaf has got a unique property of ultra-hydrophobicity^10-12. The micro and nano-scopic architecture on its surface minimizes the water droplets adhesion to the surface of the leaf. This property is also found in some other plants like taro leaf and also wings of some insects¹³.

MATERIALS AND METHODS:

Preparation of Sample:

Semi mature Lotus leaves were brought from the growing site from Hajo, (Coordinates 26°14′55″N and 91°31′32″E) Kamrup rural district. The leaves were kept in refrigerator in order to prevent it from drying. Fresh lotus leaves or refrigerated one were taken and cleaned with mild detergent washing. Cleaned leaves were put in warm water (600C) for one and half hour, so that the chlorophyll comes out of the leaf. Then the sample was dried in room temperature before proceeding to further steps, following the methodology as described by Nasri et al¹⁴.

Bio-wax extraction the leaves were cut into small pieces and immersed into chloroform measuring thrice (v/w) the amount of leaves used for 20 seconds. This was repeated thrice using fresh chloroform for the same sample. Then the solvent was filtered out and evaporated. To collect the wax remnant from the flask, a little amount of chloroform was added^15,16.

Bio wax spray on paper:

The extracted bio wax solution was put into an atomizer and sprayed on paper. The paper was left to dry before proceeding to further processes

Hydrohobicity test:

The hydrophobicity test was performed through the measurement of the time needed for 100µl of distilled water to be absorbed by the paper. The paper was placed on a glass plate (TLC plate), and secured with rubber bands. 100µl of distilled water was poured on it (paper) with the help of a micropipette, so as to avoid a forceful impingement of the water on the paper. The scale that determines the hydro-phobicity or hydro-philicity of the paper, is the time taken by the paper to absorb the water. Time recording starts when the first drop of water touches the paper and stops when the water gets absorbed by the paper. If the time recorded is more than 300 seconds, then the sample tested is said to be hydrophobic. On the other hand if the time of absorbing is less than 300 seconds, the sample hydrophilic. The sample (bio-wax coated paper) was compared with plain-non coated paper as well as only chloroforms spraying paper. The paper used for bio-wax coating was procured from market, students practice copy quality. The coated paper was also exposed to rain along with the normal uncoated paper¹⁷.

Contact angle:

To determine the contact angle of a droplet on a solid surface, a contact goniometer was used. This device allows for visual measurement of the contact angle by depositing a droplet of liquid onto the surface using a syringe. A high-resolution camera captures an image of the droplet from either a profile or side view, which can then be analyzed using image analysis software to obtain a static contact angle measurement. The contact angle is a crucial parameter in assessing the strength of the contact between a liquid and a solid surface.

The contact angle goniometer measures the contact angle of a sessile droplet using an optical system to capture the profile of a liquid droplet on a solid substrate. The contact angle θ is the angle formed between the liquid surface and the contact surface outline. This angle is used to measure the wetting ability of the solid by the liquid. A contact angle of 0° indicates complete spreading of the liquid on the surface, while a contact angle greater than 90° indicates that the surface is hydrophobic for measuring the contact angle of the bio-wax coated paper, 2µl of sessile drop of water was dropped on the paper surface. Temperature was maintained at 27°C. The paper was fixed on the slide with the help of tape. The steps were repeated 4 times^11,18.

Fourier transform infrared spectroscopy (FTIR) analysis:

Fourier Transform Infrared (FTIR) spectroscopy relies on the fact that molecules absorb light in the infrared region of the electromagnetic spectrum. The absorption of light corresponds specifically to the bonds present in the molecule, and the frequency range is typically between 4000-600 cm-1. To perform the experiment, a liquid cell is used for transmission measuring. The sample is first dried on a KBr disk and then sandwiched under another aperture plate to prevent gas bubbles from being trapped. The thickness of the sample is adjusted by inserting spacers between the aperture plates, based on the sample absorbance. The sample forms a thin liquid membrane between the two plates, and the aperture plate should be selected with the appropriate wave number range¹⁹.

To prepare the sample, approximately 2mg of the sample and 300mg of Spectroscopic Grade KBr powder are transferred to an agate mortar. The powders are ground together with an agate pestle until the sample is well dispersed and the mixture has the consistency of fine flour. The ground mixture is then transferred into the cylinder bore so that it is evenly distributed across the polished face of the lower pellet. A second pellet is inserted with the polished face towards the mixture, followed by the plunger. The die assembly is placed into a hydraulic press between the ram and piston, ensuring that the die is firmly held in the press. A vacuum tube is connected and the high vacuum pump is switched on. The die assembly is left under vacuum for approximately 2 minutes to remove air from the disk. The pressure in the press is increased to 15 lbs, and after approximately 1 minute, the pressure is released slowly. The vacuum is carefully released, and the die is removed from the press. The KBr disk is transferred to a spectrometer disk holder, being careful not to touch the faces of the disk, and checked for translucency and sample homogeneity^20,21.

RESULT AND DISCUSSION:

Hydro-phobicity test:

The hydrophobicity test showed the average time for the test was 11minutes which exceeded the 5minutes limited for hydro-phobicity. However it was also seen that the hydro-phobicity level depended on the number of coating. Also it was observed that as the plate on which the paper was fixed, on inclining, the water droplet rolls off the paper, without wetting the paper which does not happen in normal paper, as well as chloroform coated paper. The little bit of water that is not absorbed by the normal paper, on inclining, wets the paper.

Table 1: Absorbance test Run Time

Trial (min)

Sample

III

Coated (Bio wax)

Non- coated

(A) (B)

Fig. 1: (A) Non coated-the water droplet wets the paper as the glass plate is inclined

(B) Coated-the water droplet doesn’t wet the paper as the glass plate is inclined

Contact Angle:

The observed contact angle of droplets of distilled water on the taro wax surfaces is greater than 900. Three factors influence the contact angle of a liquid droplet on a surface. These are surface tension of the liquid (force of molecules), surface of the solid and the surrounding vapour. In this observation only solid surface (lotus wax-coated glass slide) was taken into account and believed to be the main factor shaping the liquid droplet onto more spherical type. It matches the expected contact angle of a hydrophobic surface²².

The result showed that with the increase in coating of the paper of the contact angle increases. Here, only for the fourth trial, double coated paper was used.

Fourier transform infrared Spectroscopy:

Like a fingerprint no two extraordinary atomic structures deliver the same infrared range. This makes infrared spectroscopy valuable for a few sorts of examination. The mid infrared range (4000–400 cm−1) is more or less separated into four districts. The way of a gathering frequency is controlled by the area in which it is found. The areas are summed up as takes after: the X–H stretching area (4000–2500 cm−1), the triple bond area (2500–2000 cm−1), the double-bond area (2000–1500 cm−1) and the fingerprint area (1500–600 cm−1).The principal vibrations in the 4000–2500 cm−1 area are because of O–H, C–H and N–H stretching. O–H stretching delivers a wide band that happens in the extent 3700–3600 cm−1

When infrared radiation was passed through the sample (lotus wax) a trace of noise was heard. An extremely broad band due to alcohol group appeared at 3431cm-1 for the sample (FTIR graph).

Fig 3: FTIR Graph of Bio Wax

From the graph the following observations were made:

Class	Group	Wave number (Cm^-1)
Alcohol	O-H	3431.24
Alkane	C-H	2918.84 2850.52
Sulfate	S=O	1380.88
Anhydrides	O-C	1045.63
Sulfur Ester	S-OR	719.03
Aromatic	C=C	1467.60
Amide	C=O	1641.15

CONCLUSION:

Surfaces are supposed to feel good to the touch and to look good for as long as possible, be easy to maintain and not be soiled by dirt, water stains. Nowadays it’s the age of nanotechnology. Although by nanotechnology the Lotus effects have been copied but to date there are only a few low cost processes which can reduce the surface structure, similar to that of the leaf that are known. Due to the cost factor it’s not at all affordable for everybody.

Although this project which is based on the extraction of the biowax from the lotus leaf has only been tried on paper, the result is satisfactory enough which gives us a hope to try this similar technique on other products.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENT:

Authors are thankful to Principal Pandu College for providing necessary facilities to carryout the work. Also thankful to Institutional Biotech Hub Pandu College supported by DBT, Govt. of India and management of Assam down town University for analytical help.

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Received on 17.04.2024 Revised on 14.08.2024

Accepted on 28.11.2024 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):2070-2074.

DOI: 10.52711/0974-360X.2025.00296

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Sample	TRIALS (in degree)
Sample	FIRST	SECOND	THIRD	FOURTH
Lotus wax	95	99	117	120