Category Archives: Stainology

Factors that Cause Loss of Quality in Essential Oils: Part I – Headspace

Does Your Oil Spoil?

Do you ever find that once you have used your essential oils a few times the liquid that remains in the bottle begins to “go bad” a great deal faster? You begin to notice, that suddenly, when you use the same bottle of essential oil, with your usual henna powder, following your usual henna recipe and mixing process, the depth and tones of the color of the stain results achieved by your henna body art is significantly reduced?

It seemed like the very same bottle
of essential oil was fine
just a few days ago;
what’s happened?


What in Essence are Essential Oils?

Essential oils, found in plants, are plant metabolites comprised of a multitude of different yet often structurally similar compounds that are categorized according to a variety of different chemical classes[1]. A single essential oil can be comprised of only a few or up to more than 100 different compounds[1][3] characterized by several key qualities:

  1. Volatility: heir ability to vaporize or evaporate far more readily than other substances at the same temperature and pressure;
  2. Lipophilicity: their tendency to dissolve in fats and oils and opposed to water-based or aqueous solutions;
  3. Molecular weight: generally does not exceed 300

Their molecular properties allow essential to be easily be separated from the plant components and tissues in which they naturally are found. While the actual role of essential oils in nature is still the subject of research and discussion, their medicinal and therapeutic properties as well as their high degree of benefit in the pharmaceutical, food, and cosmetics industries as a natural alternative to synthetic chemicals has been clearly demonstrated within the scientific community[1].


Natural Henna Body Art and the Role of Essential Oils

The henna plant, botanically known as Lawsonia inermis L., is used for body art due to its capacity to stain skin cells. When then leaves of the henna plant are harvested, dried, and crushed into a fine powder, they can be made into an paste using an acidic aqueous solution[2]. When the resulting paste is applied to the skin, the henna plant’s natural dye molecule, lawsone, can gradually migrate from the paste into the outer layers of the skin. It binds to keratin, the key proteins comprising the skin, by way of the Michael addition, creating a covalent bond between the keratin and the dye pigment[2].

As every natural henna artist knows, ‘terping’ henna paste, is an entirely natural way to achieve darker and deeper tones in the final stain that remains on the skin. ‘Terping’ refers to monoterpenes or monoterpene alcohols, a key compound comprising many essential oils. Essential oils with particularly high levels of monoterpene alcohols, such as tea tree, cajeput, eucalyptus or lavender, can be added directly into henna paste to facilitate darker stain results[2].


What is Essential Oil Quality Loss?

Quality loss, respective to essential oils, specifically refers to the chemical change of the components that cause a given essential oil to possess its beneficial properties.

The primary causes of quality loss in essential oils is their susceptibility to conversion and degradation reactions. Numerous factors exist that can bring about these two types of reactions and thus affect the stability of essential oils. The foremost of these factors being[1]:

  1. Light: UV exposure
  2. Temperature: exposure to heat
  3. Oxidation: exposure to oxygen
  4. Chemical Reaction: contact with substances that the compounds in the oil can chemically react with

The topic of this post will be the discussion the effect of oxygen, specifically headspace oxygen, on the degradation of essential oils.

canning-jar-head-space

 

Headspace is the area within a sealed container that lies above the liquid phase[1].  Headspace measurements and calculations are important for understanding and implementing accurate methodologies for the preservation of the contents of the container. Biochemical engineers Across numerous industries such as food, cosmetics, pharmaceuticals and others, use extensive mathematical calculations and knowledge of chemistry, in order to establish the exact molecular composition of this seemingly small area, and from those calculation can predict changes in composition at any given temperature and pressure.

Identifying the Signs of Quality Loss in Essential Oils

There are several qualitative indicators that can reveal that an essential oil has undergone loss of quality[1]:

  • a shift in the scent or flavor of the oil to those that are pungent and often unpleasant; 
  • change in color of the oil, often a yellowing;
  • changes in consistency of the oil such as thickening or resinification which is the formation of thick dry films that are often found on the inner cap or the neck of the bottle;

In addition to changes in physical characteristics of essential oils, some essential oils that have undergone quality loss due to oxidation have revealed skin sensitizing capability. Skin sensitization is the onset of allergic contact dermatitis or a similar irritating dermal reaction to contact with a specific substance.


The Relationship Between Size of Headspace and Rate of Quality Loss

When essential oils are used, the bottle’s cap is removed, some of the oil is poured out and the cap then replaced. A direct relationship between the size of the headspace and the rate of quality loss of the essential oil has been determined[1].

This means that the less essential oil there is remaining in the bottle, the faster it will tend to spoil and thus be unable to produce the same results when used in henna paste. NOTE: This is true irrespective of the volume of the container, i.e., it is as true for a large vat as it is for a vial with similar physical proportions.

Solution & Best Practice:

Keep a collection of different sized bottles with capacities to store different volumes of liquids in order to significantly extend the life of your essential oils and their stain-producing capabilities. When the oil contained in a given bottle begins to drop below 75% full, transfer into a smaller bottle should be made to minimize the headspace.

These UV-proof essential oil storage bottles from Anandaapothecary.com are as good as it gets. The are made of Swiss UV-proof glass and can come with a whole bunch of useful accessions that you can read about on their website: https://www.anandaapothecary.com/ananda-apothecary-news/state-of-the-art-storage-bottles-for-your-essential-oils

uv-proof-bottles-300x216

They are pretty amazingly reasonable priced for being just about the best storage bottles that I have come across, and If anyone, say, wants to get me a gift for no particular reason… umm these bottles are just about tops on my list.

A second best alternative within an affordable price range are a collection of the standard metal essential oil storage bottles, used mostly for storage of large quantities of essential oils ranging from 250 mL to more than 1L.

pack2

These are outstanding for long-term storage. Be sure, however, to get the bottles with the airtight plug on top as some companies just sell bottles with a screw-on cap. The airtight plug makes a significant difference in the preservation of the oil.

The downside to these bottles, however, is the potential for the essential oil to interact with some of the metallic compounds that make up the bottle. I hope to make this issue the topic of a future blog post.

The ideal bottle type for storing essential oils:

  • is made from glass 

  • has an air tight (or close to it) cap

  • is dark in color, protecting the oil from light essential oil storage box

 

Best Practices for Storing Essential Oils Include:

Keep stored bottles in a cool, dry, dark place for best results.

My favorite storage container: the wooden slotted essential oil storage box with a clasped lid and a carrying handle–have oil will travel.

 


So, there it is: the problem, the cause, and the solutionall wrapped up in a few paragraphs.

If, however, you are a henna nerd and possibly possess henna OCD ( not yet in the DSM V but I am still sure that I can be diagnosed with it) feel like you absolutely must understand the specifics of the science behind just why this happens, the rest of this post is for you. 


1.0 Liquid-Vapor Equilibrium 

Picture7

When your essential oils are contained within a tightly closed jar, they can be said to form what is known to biochemical engineers as a closed liquid-vapor system[4].

A closed liquid-vapor system, respective to essential oils, is simply an air-tight vial or jar that contains some essential oil and some air at the top, i.e., headspace.

 

A closed liquid-vapor system, such as a glass bottle containing an essential oil reaches and settles in a state of liquid-vapor equilibrium. Liquid-vapor equilibrium is simply the preferred state of the matter contained within the system, and it is a function of temperature and the internal pressure of the container[4].

So, at any given temperature and internal pressure the distribution of species (i.e., different chemical components) within the liquid and vapor phases in the closed system reaches a constant value that is measured in moles of species in each phase [4].

Rosemary-Essential-Oil.jpg

The total pressure of the vapor phase of the system i.e., the headspace is the sum of the partial pressures of each chemical species within the system. Knowing that essential oils are comprised of different combinations of several compounds that possess the characteristic of volatility (high tendency to vaporize) the total pressure in the headspace of the essential oil bottle is then the sum of the pressures exuded by each species within the oil. This will be important later in understanding essential oil spoilage.


2.0 Changes in the System 

Picture9.jpg

When the essential oil bottle is opened and some liquid poured out–say, to be added to a henna paste mixture–and the cap resealed , the closed system (i.e., the bottle) will reach a new state of vapor-liquid equilibrium because the volume of liquid as well as the size of the headspace have both changed.

Picture3.jpg

The system will now tend towards a new state new equilibrium that will be a function of how many moles of substance are left in the liquid phase and vapor phases. When the lid is replaced and the system re-sealed:

  • some of the volatile essential oil molecules, due to their tendency to vaporize, will vaporise or evaporate and leave the liquid phase passing into the vapor phase and occupying the bottle’s headspace and increasing the vapor pressure of the headspace;
  • as the pressure in the vapor phase or the headspace increases, some of the oxygen in the headspace will be “pushed out” of the vapour phase and into the liquid phase, i.e., the essential oil,

Now, with more oxygen dissolved in the liquid phase of the essential oil, oxidative damage is occurring at a faster rate. It can be seen from this example that the greater the headspace the faster the rate of oxidation in the liquid, i.e., essential oil spoilage.

 


Picture11.jpgTo Summarize:

After some of the essential oil has been poured out and the bottle resealed:

  1. less liquid remains in the bottle;
  2. there is an increase in headspace;
  3. at the precise point in time of sealing the bottle, the headspace or vapor phase is comprised of the air that entered the bottle when the cap was removed and liquid was poured out;
  4. due to the tendency of components that comprise essential oils to vaporize (volatility), the species from the liquid phase will begin to evaporate and enter the vapor phase;
  5. as species enter the vapor phase, the pressure of the vapor phase increases;
  6. as the pressure of the vapor phase increases due to vaporization of the volatile components oxygen from the air in the vapor phase is “pushed out” by the pressure and dissolves into the liquid phase (i.e., the essential oil)
  7. when the system reaches equilibrium at a constant temperature, the total pressure of the vapor phase, at any given time is equivalent to the partial pressures of each of the species within the system;
  8. with a greater amount of oxygen dissolved into the essential oil, the rate of oxidation or loss of quality of the essential oil due to oxidative damage is ingreased;

 


3.0 Effects of Temperature

The specifics of temperature as a cause of essential oil spoilage as well as the mechanism by which oxidization causes loss of quality in essential oils will be saved for the topic of future blog post, however, given the information we have now it can be understood why an increase in temperature affects an essential oil causing it to loose quality at a faster rate:

  1. the higher the temperature the greater the kinetic energy possessed by the molecules in the system;
  2. the greater the kinetic energy possessed the greater the tendency for the volatile compounds in the liquid phase to vaporize, leaving the liquid phase and passing into the vapor phase
  3. As more molecules enter the vapor phase the pressure within the headspace increases
  4. The increase in headspace pressure forces some of the less volatile compounds such as oxygen molecules within the air to dissolve into the liquid phase
  5. Once the liquid phase contains more oxygen the rate of oxidative damage is greater;

So a higher temperature causes a higher rate of vaporization of the liquid phase, which in turn causes an increase in pressure of the vapor phase and results in more oxygen dissolving into the essential oil.


4.0 Raoult’s Law & Henry’s Law 

The rates of quality loss of essential oils during the manufacturing process is one of the topics of concern of biochemical engineers functioning in quality assurance roles in industry. Rates can be is can specifically calculated given knowledge of the specifications of the manufacturing process as well as the precise composition of the essential oil, usually obtained through high performance liquid chromatography (HPLC)

Calculations are concerning the amounts of species in both the liquid and the vapor phases can be made using Raoult’s Law or Henry’s Law. Raoult refers to François-Marie Raoult who discovered the thermodynamic relationship between gases and liquids in a closed system in 1887.  Henry, of Henry’s Law also refers to similar but distinct thermodynamic gas law formulated by British scientist William Henry in 1803[4].

Both laws state that in a closed system (e.g., an airtight bottle) that the distribution of substances between gaseous and liquid phases are determined by specific relationships that exists between the partial pressure of a volatile liquid–that is, the one that can evaporate–and the molar composition of the component in the mixture. Both gas laws are the subject of vapor-liquid equilibrium.


 

5.0 Conclusion

In conclusion, of the best practices for preservation of essential oils and prevention of quality loss is keeping a set of numerous containers varying in size. When the liquid levels begin to decrease, storage in a smaller container prevents an increase in the rate of quality loss due to oxidation. 


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Patreon-logo-16x9

 


References:

[1] Turek, Claudia, and Florian C. Stintzing. “Stability of Essential Oils: A Review.” Comprehensive Reviews in Food Science and Food Safety, vol. 12, no. 1, 2013, pp. 40–53., doi:10.1111/1541-4337.12006.

[2] Gallo, Francesca R., et al. “Henna through the centuries: a quick HPTLC analysis proposal to check henna identity.” Revista Brasileira de Farmacognosia, vol. 24, no. 2, 2014, pp. 133–140., doi:10.1016/j.bjp.2014.03.008.

[3] Handbuch des Arznei- und Gewürzpflanzenbaus. Band 1 Grundlagen des Arznei- und Gewürzpflanzenbaus I, Blitzke T., 2009

[4] Felder, Richard M., et al. Elementary principles of chemical processes. Wiley, 2017.

[5] Hüsnü K., Başer C., Demirci F. (2007) Chemistry of Essential Oils. In: Berger R.G. (eds) Flavours and Fragrances. Springer, Berlin, Heidelberg

 

Hennapedia: The More You Know…

Achieving excellent results using all-natural or organic henna powder does not occur by chance. I often hear those who fail to get a deep bold and long-lasting stain blame the product or their own “body chemistry,” making excuses for their lackluster stain results.

Likewise, all too frequently I encounter individuals who observe incredible results henna stain results from an all-natural henna powder who, not yet having achieved similar results, become frustrated and convince themselves and others that the henna must have been adulterated or that the individual just has the “right body-chemistry” because such results “just can’t be real.”

 

IMG_20160819_201624Paste ON24x8

 

Perhaps in the rarest of cases this may be true, however, for the overwhelming majority of such situations, it’s what you know. One does not have to be a scientist to get an epic henna stain, however, possessing a basic knowledge of how skin and hair are comprised, the basic reaction mechanisms involved in henna staining, and some several other details can take your henna stain from pale orange to deep rich tones that can even reach a black color …naturally.

The Hennapedia Initiative is a project that combines two of my greatest passions in life: all-natural henna art and scientific research. The objective is to ultimately be an ever-growing comprehensive one-stop source of open-source information for all things henna: from mixology to stainology to even starting a henna business.

The Hennapedia vision is to allow new artists to avoid the years of trial and error and frustration that many (including myself go through) and to allow the pros to troubleshoot any issues that may come up in a matter of moments.

As a biochemical engineer, I have an almost obsessive love for getting down to the details when it comes to henna, indigo, and other ayurvedic herbs. Hennapedia is an opportunity to share the knowledge that I am learning as I go so that you don’t have to go through the work that I do. Hennapedia seeks to create an ever-growing knowledge base that serves as an easy reference tool for anyone interested in the topic of henna and plant-based dyeing as well as other relevant related subject matter.

The only things stopping me is: I need your help. Research is costly, and time consuming. Equipment is prohibitively expensive, and time waits for no one. I ask you to support my work on patreon. You can make a difference for as little as $1 per month.

My dream is to be in a position where I can work for you full time, providing accurate, cited, peer-reviewed science in response to your henna questions. There are some incredible scientists out there doing phenomenal research, there is just no real central source of up-to-date information.

Consider the value you can add to your henna business for as little as $1 a month and please become a Patron of Hennapedia …because the more you know the more we grow.Patreon-logo-16x9.

 

 

Tutorial: Henna Precare & Preparation

Did you know that What you do to your skin just before you get your henna applied Stands to significantly affect the quality of your henna stain?


What you do in preparation for the application of a henna design on your skin–called precare–can dramatically affect your final results. Preparation and precare stand to affect numerous aspects of the final henna design by:
  • increasing the darkness of stain color;
  • increasing the depth of stain tone; and
  • increasing the lifespan of the henna design.
In order to understand why this is the case, it is necessary to understand exactly how henna stains the skin on a molecular level.

First, What Exactly is Human Skin & How Does it Work

skin

A Cross-Section of the Epidermis

The stratum corneum, the outermost layer of the skin is comprised of dead skin cells with no nucleus , nor cytoplasm. These cells are merely sacs filled with keratin proteins and no cellular function. The stratum corneum contains within it 9 layers of keratinocytes; the cells in the topmost layer continuously flake off to be replaced with the new younger cells beneath.
(OpenStax, Anatomy & Physiology, 2016)

The reason that dyeing the skin with henna paste works so well is that lawsone (the active pigment in henna paste) can form bonds with the keratin molecules on the outer layer of skin (the stratum corneum). Staining is highly effective due to the abundance of available keratin: the primary remaining component of a skin cell after cell death (OpenStax, Anatomy & Physiology, 2016).

So, How Exactly Does Henna Work?

Lawson molecules are the dye pigments that brings about a henna stain on the skin or hair. While free lawsone does not occur naturally within the henna plant, it is derived from precursors called hennocides that are found within the leaf. When the powdered leaves of the lawsonia inermis plant (henna plant) are soaked in an acidic aqueous solution, ionized lawson carrying a negative charge is formed, In addition, when long keratin molecules come into contact with an aqueous acidic solution, the amino groups on the keratin molecule accept protons from the acid and thus become positively charged (Amro et al. 1993).

The now ionized and free lawsone molecules carrying a negative charge can now bond with the ionized amino groups sites on the long chain keratin molecule which carry a positive charge. The key to bonding, however, is proximity. Lawsone ions must come within a very close proximity to the keratin molecule’s amino sites in order to successfully begin to share electrons thereby forming a chemical bond (Amro et al. 1993).

How Henna Works

Process Diagram:

  1. Bound hennocides exist within the leaf of the lawsonia inermis plant;
  2. When contacted with an aqueous acidic solution, the hennocides undergo several chemical changes the product of which are free lawsone ions, possessing a negative charge, existing within the wet henna paste.
  3. When applied to skin, the acidity of the aqueous solution from the moisture contained within the paste ionizes the amino groups along the molecular keratin chains contained within the outer dermal layer of dead skin cells.Lawson ions within close proximity to positively charged amino groups are able to form covalent bonds with one another.As the moist paste remains on the skin, dyeing is taking place, and the lawson molecules are becoming permanently affixed to the keratin chains: your henna tattoo is staining your skin.

The Type of Chemical Bond

334px-Covalent.es

A Methane Molecule Depicting Covalent Bonding

The type of chemical bond formed between the lawsone and the keratin molecules is called a covalent bond . A covalent bond is a permanent chemical bond that is formed by the sharing of electrons between atoms.

Because lawsone molecules are now permanently affixed to keratin chains via their amino groups, the stain that results from henna is actually considered a permanent stain. (Amro et al. 1993)

 

If a Henna Stain is Permanent, Why Does it Fade After Time?

The reason a henna stain on skin fades is not due to the bonds between the lawson molecules and the keratin amino groups breaking, nor is the dye pigment losing its colour. The reason that henna tattoos fade is that the skin cells that are dyed are those on the upper-most layer of skin, the stratum corneum. As discussed these skin cells are in a constant process of being shed in order to be replaced with younger cells underneath from the deeper layers of skin. (Gallo et al. 2014). Therefore, in essence your henna stain is being shed, it is not fading away.


skin-cells-life-cycle
Did you know that the human skin
regenerates itself entirely every
45 – 48 days? (Hajime Iizuka, 1994).
That means that we have an
entirely new skin every six to
seven weeks.

 


Now that we know the Process by which lawsone interacts with the skin to form a “henna stain,” what can we do to engineer a optimal natural method that will Prepare the skin for the best henna stain results?

First, Let’s Review What we Know:

  1. When contacted with acidic aqueous solution both lawsone and keratin become ionized and thus become available for bonding with one another (Amro et al. 1993);

     

  2. Bonding can only occur when lawsone ions are within a close proximity to the keratin’s ionized amino groups (Amro et al. 1993);

     

  3. The dead skin cells that make up the outermost layer of the skin (the stratum corneum) are comprised mostly of keratin and are in a constant process of regeneration, being replaced with new ones from deeper layers within the skin (Gallo et al. 2014);


Engineering an Optimized
Precare Process

Identification of the Factors Preventing Bonding

  1. As discussed, the dead skin cells at the very surface of the skin are at the end of their lifespan as part of the epidermis and are as a result flaking off to be replaced with the younger cells underneath. As a result, dyeing these cells with henna will result in a stain that quickly fades as the already aged cells flake off.
  2. In addition, the surface of the skin, as a result of these cells is more rough and uneven causing it to become more difficult for the lawsone ions in the paste to gain the necessary close proximity to the ionized keratin amino groups.
  3. Another factor affecting the ability of the lawsone ions to gain proximity to the ionized functional groups on the keratin strands is the normal buildup that occurs on skin consisting of natural oils, environmental contact, and any skincare products applied such as moisturizers or creams. Oily substances, such as hand lotions or the skin’s natural oils are very effective at forming a barrier between skin cells and lawsone applied in the form of henna paste.

Green-Check-Mark

 

Conclusion

  1. To obtain best results skin cells that are reaching the very end of their lifecycle should be removed so as to prevent a short-lived henna stain;
  2. It is of critical importance to remove any substances that may be coating the surface of the skin or acting as a barrier preventing lawsone ions to be able to come into close proximity to the amino groups on the keratin cell before henna application;

Solution:
Developing a Precare Process

*NOTE: This method is not for people who have sensitive skin, naturally dry skin, or eczema. See suggested alternate method, please use caution and avoid any steps that may cause skin irritation.

Step 1:
Soak hands in warm Salted water

Picture7.jpg
  • Fill a clean glass bowl with warm water add 1-3 teaspoons of salt. Soak hands for 10 – 15 minutes. The warm water will soften your skin and the salt will draw moisture out of the cells comprising the most external layers of skin making them more easy to remove by exfoliation.

Step 2:
Exfoliate

aid1336502-v4-900px-Use-a-Pumice-Stone-Step-10-Version-2
  • Using a pumice stone or another exfoliating tool of choice, rub the area of skin where the henna design is to be placed well.
  • Rinse stone and area of skin in the warm water and repeat.

NOTE:

DO NOT use exfoliating creams, scrubs, or similar products. Such products will leave an oily residue on skin forming a barrier preventing staining. Best exfoliation methods are those that involve a firm tool such as stone or metal scrubber that is not synthetically made from polymer-based materials that can absorb oils and contaminants

195048_Sunlight_Lemon_950mL20171127

Step 3:
Soap Strip

    • Add a generous amount of liquid dish soap onto your palm.
    • Lather the area of skin to be tattooed and rub the area well–you can use your pumice stone to scrub the area if your skin is not too sensitive.Picture11.png

 

  • Do not use a liquid dish soap that contains a skin moisturizer or that claims to be  “soft on hands.” The purpose of this step is to temporarily strip your hands of natural oils so that lawsone ions can easily come into close proximity with the skin’s keratin.
  • Sunlight Lemon is an excellent choice for this step as it contains no ingredients that will add oils or residues to your skin, and it’s capacity to remove oils and grease is excellent.

 

 

 

Step 4:
Dry Hands with a Paper Towel

 

  • Dry off your hands with a clean paper towel. Do not use a bathroom towel or a dish towel as they can retain oils and other substances from previous usage–even if recently laundered–that can leave contact residue on your now prepared skin.
  • Avoid using toilet paper as it leaves a dusty residue.

Step 5:
Apply Henna

Your hands should be noticeably smoother and drier. Your skin may feel a little tight as the oils that naturally lubricate it have been temporarily stripped away. This feeling is not a sign of harm and it should dissapear after a few hours.
Once you have completed the prevcare process, do your best not to touch anything or to allow the area of skin when henna application is to occur to come into contact with any substance so as to avoid the risk of adding residue to the skin.
  • Be sure to get your henna applied as soon as possible after preparation and precare have been completed, before natural oils return to the skin and contact with environmental residues occurs.
NOTE: For sensitive skin, use your own judgement and experience to determine if ANY precare is appropriate for your skin type. If unsure it is best to avoid.

 References

  1. OpenStax, Anatomy & Physiology. OpenStax CNX. Feb 26, 2016 http://cnx.org/contents/14fb4ad7-39a1-4eee-ab6e-3ef2482e3e22@8.24

  2. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Epidermis and Its Renewal by Stem Cells. Available from: https://www.ncbi.nlm.nih.gov/books/NBK26865/

  3. Iizuka, Hajime. “Epidermal architecture that depends on turnover time.” Journal of Dermatological Science, vol. 10, no. 3, 1995, pp. 220–223., doi:10.1016/0923-1811(95)00407-j.

  4. Gallo, Francesca R., et al. “Henna through the centuries: a quick HPTLC analysis proposal to check henna identity.” Revista Brasileira de Farmacognosia, vol. 24, no. 2, 2014, pp. 133–140., doi:10.1016/j.bjp.2014.03.008.
  5. Amro, B. I., et al. “A Quantitative Study of Dyeing with Lawsone.” Journal of the Society of Cosmetic Chemists, no. 45, 1994, pp. 159–165.
  6. França, Simone Da, et al. “Types of Hair Dye and Their Mechanisms of Action.” Cosmetics, vol. 2, no. 2, 2015, pp. 110–126., doi:10.3390/cosmetics2020110.
  7. Bhuiyan, M.a. Rahman, et al. “Color and chemical constitution of natural dye henna (Lawsonia inermis L) and its application in the coloration of textiles.”
  8. OpenStax, Chemistry. OpenStax CNX. Jun 20, 2016 http://cnx.org/contents/85abf193-2bd2-4908-8563-90b8a7ac8df6@9.311.