Field of the invention

The present invention provides powdered plant material which has been fortified with a metal such as zinc. This may be used in the treatment of metal deficiencies and related disorders.

Background

Nutrient deficiency is a problem faced across the world, with the diet of many of the world’s population lacking one or more essential nutrients. This is particularly the case for people in rural and deprived areas, where the dominant part of their diet may be a single stable food such as rice. Nutrient deficiency can result in issues such as poor immune system health, heart disease, anemia, blindness, cancer and early death.

Nutrient deficiency can be remedied through means such as diversification of diet, mineral supplementation, food fortification, or increasing the concentrations and/or bioavailability of mineral elements in produce (biofortification).

Diversification of a persons’ diet may be a solution to this issue. However, such diet diversification is simply unaffordable and/or unavailable to many, particularly those in rural and deprived areas who are at risk of malnutrition. Similarly, mineral supplementation may not be an affordable or practical option for many. Fortification of food nutrients can also be difficult.

Biofortification involves increasing the nutrient content of foods or crops as they are cultivated, and may be a good option for combating nutrient deficiency because it can be affordable and widely available. This is particularly the case where fortified crops are staple foods, meaning that no additional costs to the consumer are incurred, and no change to their diet or habits are needed.

One particular deficiency is zinc deficiency, which is known to be a major risk factor for human health and cause of death globally. Zinc deficiency may occur in humans where the food that they consume is also deficient in zinc, which may be a result of zinc-deficient soil.

It would be desirable to provide new ways to cure and/or prevent nutrient deficiency, such as zinc deficiency. Summary

In one aspect, the present invention provides powdered Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof, which have been fortified with a metal, such as zinc.

The present invention also provides a method of fortifying Salvia fruticosa Mill, and/or Aloysia cotriodora Palau, comprising applying metal oxide nanoparticles (such as zinc oxide nanoparticles) to Salvia fruticosa Mill and/or Aloysia cotriodora Palau.

The present invention also provides a method for making metal oxide nanoparticles, such as zinc oxide nanoparticles, the method comprising:

(i) obtaining a plant extract from Salvia fruticosa Mill, and/or Thymbra capitata (L.) Cav.; and

(ii) adding a metal salt.

The plant extract of Salvia fruticosa Mill, and/or Thymbra capitata (L.) Cav. may be obtained by harvesting plant material from Salvia fruticosa Mill, and/or Thymbra capitata (L.) Cav.; optionally drying the plant material; adding water and heating to form an aqueous solution comprising the plant extract; optionally filtering the solution to obtain an aqueous plant extract; and optionally removing the water to form a dried extract.

Figures

Figure 1 shows the concentration of zinc in foliage samples of fortified Salvia fruticosa plants after exposure to various treatments.

Detailed description

Nutraceutical

In one aspect, the present invention provides powdered (or particulate) Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof, wherein the Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof have been fortified with a metal, such as zinc.

The invention also provides a composition comprising powdered plant material from (i.e. obtained or derived from) a plant selected from the group consisting of Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof, wherein the plant has been fortified with a metal. That is, the plant has been fortified with a metal before being formed into powdered plant material (i.e. before harvesting).

Put another way, the invention provides powdered and metal-fortified (e.g. zinc-fortified) plant material of (i.e. obtained or derived from) Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof.

Put yet another way, the invention provides plant material obtained or derived from Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof, wherein the Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof have been fortified with a metal, such as zinc, and wherein the plant material is in the form of a powder or particles.

The composition may additionally comprise one or more additional components or compounds, for example to change or improve the taste of the composition. These additional components or compounds may include any known food additives and flavouring substances, preferably those which are natural and approved for use in food or beverages.

In one embodiment, the composition therefore also comprises powdered plant material from a second plant which is different to the first plant (the first plant being Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof). The second plant material may change or improve the taste of the composition compared to when the second plant material is absent. Suitable plants for providing the second plant material include, for example, plants of the families Lamiaceae and Rutaceae, as well as Zingiber officinale and Aloysia citrodora. In another embodiment, the composition additionally comprises a compound providing a taste or health benefit, such as vitamin. Suitable vitamins include any known vitamins, such as Vitamin A, B, C, D, E, K, etc.

As used herein, the term “plant material” means material which is obtained from or derived from a plant. This could include the whole plant or a portion thereof. Suitable plant material for use in the present invention includes, but is not limited to, leaves, roots, rhizomes, bark, fruits, flowers, or combinations thereof. Preferably, the plant material comprises, consists essentially of, or consists of, one or more leaves. The plant material may be harvested from the plant and then formed into a powder using methods which would be known to the skilled person (e.g. grinding). The powdered plant material used in the present invention is formed directly from the plant material. That is, the plant material is formed into a powder (e.g. by grinding) without, for example, forming a liquid (e.g. aqueous) extract of the plant material and then spray drying the extract. As used herein, the term “powdered” or “particulate” plant material does not therefore include power or particles formed from a plant extract (e.g. by spray drying), at least for the first plant material. Rather, the powder or particles of the first plant material comprise the actual harvested plant material. If present, a second plant material may optionally be derived from a plant extract.

As used herein, the term “fortified plant material” refers to plant material obtained from or derived from a plant which has been fortified in the manner described herein. The fortification processes described herein are all biofortification processes. That is, the plant is fortified before harvesting, i.e. biofortified. Thus, a reference herein to “fortified plant material” is a reference to “biofortified plant material”.

The powdered plant material (i.e. powdered plant material of Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof) is designed to be orally administered to a human, or to be added to a foodstuff or beverage which is to be consumed or ingested by a human, in order to treat or prevent a nutrient deficiency, and in particular a metal deficiency, such as zinc deficiency, or related disorders.

Thus, in one aspect the invention provides powdered plant material as described herein, for use in treating or preventing a metal deficiency or related disorders, preferably a metal deficiency. In this case, the metal deficiency to be treated is deficiency of the metal with which the plant has been fortified. Thus, in one aspect the invention provides powdered plant material as described herein, wherein the plant(s) (from which the plant material is derived) have been fortified with zinc, for use in treating or preventing zinc deficiency.

In another aspect, the invention provides a method of treating or preventing a metal (e.g. zinc) deficiency in a person, the method comprising orally administering powdered plant material as described herein, i.e. powdered plant material from Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof, which have been fortified with the metal (e.g. zinc).

The powdered plant material (i.e. powdered plant material from Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof) can be orally administered in dried form, or after being dissolved or being added to a foodstuff or beverage which is to be consumed or ingested. For example, the powdered plant material could be incorporated into a foodstuff or beverage to be consumed. Preferably, at least a portion of the powdered plant material is dissolved (e.g. in water) to form a beverage which is then consumed (i.e. orally administered).

Importantly, the fortified plant material itself (i.e. the powdered plant material from fortified Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof) should be ingested to achieve the maximum effect of treating or preventing metal deficiency. The plant material can therefore be added to a foodstuff or beverage which is to be consumed or ingested.

By way of example, the fortified plant material can be used like a macha tea, i.e. the powdered fortified Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof may be dissolved in a liquid and then the resulting liquid (i.e. beverage) consumed. This contrasts to a traditional tea-making process, in which non-powdered forms of fortified plant material (e.g. leaves, which may be whole or shredded) are contacted with a (usually hot) liquid (usually water) and then the liquid (but not the plant material itself) is consumed. In contrast, by consuming or ingesting the whole of the fortified Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof, the benefit of the fortification is better achieved.

By way of example, it has been found that when Salvia fruticosa has been fortified with zinc, the levels of zinc in hot or boiling water which has been contacted with non-powdered Salvia fruticosa (specifically whole Salvia fruticosa leaves) is still less than 10 ppm due to the immobilization of zinc in plant tissue. As such, the use of non-powdered Salvia fruticosa (e.g. Salvia fruticosa leaves like a classical tea, for example in teabags) will not be sufficient to contribute to zinc intake by the consumer. Instead, it has been found that the consumption of the Salvia fruticosa via the powdered Salvia fruticosa extract of the present invention is necessary.

In order to achieve the effect discussed above, it is desirable for the fortified Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof to be in powdered or particulate form.

The powdered plant material (i.e. powdered Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof) used in the present invention may have a particle size between about 1 and about 10 pm, preferably between about 5 and about 50 pm, more preferably between about 15 and about 20 pm. The particle size may be measured using methods known in the art, for example by using sieves having known hole sizes.

That is, as used herein, the term “powdered” or “particulate” preferably means particles having a size (i.e. diameter or longest dimension) of from about 1 and about 10 pm, preferably between about 5 and about 50 pm, more preferably between about 15 and about 20 pm. As mentioned above, the particles are not formed from a plant extract (e.g. by spray drying), but are instead formed directly from the plant material.

The metal (e.g. zinc) content of the powder may range from about 1000 to about 6000 ppm, such as from about 2000 to about 5000 ppm, preferably from about 3500 to about 4500 ppm.

The powdered plant material discussed above comprises a powder formed from Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof, which has been fortified with a metal such as zinc, preferably using the methods described herein. The powder may be formed by harvesting the plant material (preferably the leaves) which has been biofortified, optionally drying the plant material, and then grinding the plant material to form a powder.

Thus, in one aspect the invention provides powdered leaves of Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof, wherein the Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof have been fortified with a metal.

The invention also provides powdered Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof, formed by grinding the leaves obtained from Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof which have been fortified with a metal.

The present invention also provides a method for forming a biofortified powdered plant material (e.g. the material described above), the method comprising fortifying a plant selected from the group consisting of Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof, with a metal (e.g. zinc); harvesting plant material (e.g. one or more leaves) from the plant(s); optionally drying the plant material; forming the plant material into a powder (e.g. by grinding).

The plant(s) may be fortified by applying metal oxide nanoparticles to the plant(s). This type of process is a bio-fortification process, and is described in more detail below. Due to the bio-fortification process, the metal (e.g. zinc) content of the plant(s) will be higher than the metal content of an otherwise equivalent plant which has not been biofortified.

Thus, in one aspect the powdered plant material has a higher metal content than powdered plant material from an otherwise equivalent plant which has not been biofortified. As discussed further below, the content of other components of particular interest (such as p-Cymene, 1,8-Cineol, a-terpineol, linalool, trans-thujone, cis-thujone, camphor, p- caryophyllene, y- terpinene, borneol) can also be affected (e.g. increased or decreased) by the biofortification process.

Thus, in one aspect the powdered plant material has a higher content of one or more of p- cymene, 1 ,8-Cineol, a-terpineol, linalool, trans-thujone, p-caryophyllene, and y- terpinene, than powdered plant material from an otherwise equivalent plant which has not been biofortified.

Alternatively and/or additionally, the powdered plant material may have a lower content of one or more of cis-thujone, camphor and borneol than powdered plant material from an otherwise equivalent plant which has not been biofortified.

Preferably, the plant is selected from the group consisting of Salvia fruticosa Mill., Aloysia cotriodora Palau, and combinations thereof.

More preferably, the plant is Salvia fruticosa Mill.

Bio-fortification

The present invention also provides a method of fortifying Salvia fruticosa Mill, and/or Aloysia cotriodora Palau, comprising applying metal oxide nanoparticles to Salvia fruticosa Mill, and/or Aloysia cotriodora Palau.

As used herein, the term “nanoparticles” means particles having a diameter (i.e. the longest dimension) of from about 1 to about 700 nm. In some embodiments, the nanoparticles have a diameter of from about 1 to about 500 nm, such as from about 10 to about 500 nm, or from about 1 to about 100 nm.

This method is a biofortification process. Biofortification is a process by which crops can be modified to increase their nutritional value. This can be done either through selective breeding or through genetic engineering. Biofortification differs from ordinary fortification because it focuses on making plant foods more nutritious as the plants are growing, rather than having nutrients added to the foods after harvest and/or when they are being processed. In the present invention, the biofortification is achieved simply by applying metal oxide nanoparticles to the foliar of Salvia fruticosa Mill, and/or Aloysia cotriodora Palau, for example by spraying the leaves of the plant(s). The nanoparticles are readily absorbed by the plants after spraying.

The nanoparticles are preferably applied in the form of a solution comprising nanoparticles and a carrier or solvent (preferably water). The nanoparticles are preferably present in the solution at a concentration of from about 0.1 g/L to about 5 g/L, more preferably from about 0.5 g/L to about 2.5 g/L, and most preferably about 1 g/L.

The solution comprising nanoparticles may be formed by mixing the nanoparticles with a carrier, preferably water. Ultrasound may be applied to ensure the mixture is homogenised. For example, zinc oxide nanoparticles may be exposed to a probe sonicator (220 Volts, 4 Amps, power 600 watts 50/60 Hz) for 20 minutes with a step of 1.5 minutes of ultrasound exposure and 1 minute "rest".

The nanoparticles are applied after the plants have grown to maturity, and during the preflowering phenological stages plants may be exposed to two rounds of foliar spraying, specifically during the second and the fourth weeks before harvesting. No additional metals are given or applied to the plants.

After harvesting, samples of foliar tissue may be air-dried at room temperature (i.e. 25 °C) in dark conditions for about 10 days.

Preferably, the metal oxide nanoparticles are zinc oxide nanoparticles.

The metal oxide nanoparticles may be formed using the (biosynthetic) methods described herein. Using the nanoparticles formed using a biosynthetic process, such as that described herein, is preferable because this avoids potential toxicity issues with chemically-derived nanoparticles, which could ultimately be ingested if applied to the plant.

Method of making metal oxide nanoparticles

The present invention also provides a method for making metal oxide nanoparticles, which may be used in the bio-fortification processes described herein. Nanoparticles are often synthesized using conventional physical or chemical techniques. These can include sputtering, milling, chemical reduction, etc. However, an issue with these conventional techniques is that they can be expensive and/or environmentally hazardous due to the use of potentially dangerous chemicals.

More recently, biological methods for forming nanoparticles have been used, and the present invention makes use of such biological methods. Such methods are generally more environmentally friendly than previously used physical or chemical techniques. The biosynthesis of metal oxide nanoparticles such as zinc oxide nanoparticles is also relatively easy, effectively involving just a single step.

In the biosynthesis process, a plant extract is used. Said plant extract may be formed from plant material, including but not limited to leaves, roots, rhizomes, bark, fruits, flowers, and combinations thereof.

The biosynthesis process involved the collection or harvesting of plant parts or plant material (such as one or more leaves), after which the plant material may be washed with water, for example in order to remove debris and other unwanted materials. The plant material may then be either dried or ground to form a powder, or used directly to obtain an extract. For example, the plant material may be chopped into small pieces or ground to form a powder and then heated in water or another solvent (e.g. ethanol) to obtain a plant extract.

Alternatively the plant material may be heated in water or another solvent (e.g. ethanol) to obtain a plant extract without first drying the plant material and/or forming a powder.

Once the extract is obtained, a metal salt is added to the plant extract and metal oxide nanoparticles are formed.

Importantly, there is no requirement for external chemical stabilisers or reducing agents to be added. Rather, the plant extract is simply mixed with the metal salt solution. Biochemicals present in the plant extract acts as reducing agents as well as stabilising agent for the synthesis of the nanoparticles.

The synthesis of the nanoparticles can be monitored using various techniques, including by visual observation or by using UV-vis spectrometry. The synthesized nanoparticles can be extracted from the solution by known techniques, such as centrifuging. The separated nanoparticles can then be washed, for example with water. Since no additional chemicals are needed, another advantage of the biosynthetic process is that the nanoparticles can be produced free of impurities which might otherwise result from the production process. Additionally, the nanoparticles can be produced quickly and cheaply, and on a large scale. For example, the method does not require expensive equipment or precursors.

In the present invention, the biosynthetic process makes use of Salvia fruticosa Mill, and/or Thymbra capitata L.) Cav.

Thus, the biosynthetic method of the present invention comprises:

(i) obtaining a plant extract from Salvia fruticosa Mill, and/or Thymbra capitata L.) Cav.; and

(ii) adding a metal salt to the plant extract.

The result of this process is metal oxide nanoparticles, as discussed above.

Step (i)

The plant extract is obtained, derived or formed from plant material which may be selected from leaves, roots, rhizomes, bark, fruits, flowers, and combinations thereof. Preferably, the plant material comprises one or more leaves (of Salvia fruticosa Mill, and/or Thymbra capitata(L.) Cav.).

The plant material is harvested and then may be washed to remove debris and other unwanted materials.

The plant material may then be dried, although this is not essential. This drying step may involve heating the plant material for a period of time, but can also involve simply leaving the plant material at ambient conditions and allowing any moisture to evaporate. For example, this drying step may comprise air-drying the plant material at 25 °C (i.e. room temperature) for a period of 24 hours or more, such as about 5-15 days, preferably about 8-12 days.

After any drying step, the plant material may then be formed into a powder (e.g. by grinding, such as using a pestle and mortar). However, this step is also not essential, and the plant material can be used immediately after harvesting and any washing step(s). The plant material, which may have been dried and/or ground to a powder, is then mixed with water and heated to form an aqueous solution comprising the plant extract. This step preferably comprises heating a mixture of water and plant material to between about 30 °C and about 100 °C, preferably between about 50 °C and about 70 °C, for between about 10 and 60 minutes, preferably between about 15 and 30 minutes. The resulting mixture may then be allowed to cool to room temperature. This step may be carried out at atmospheric pressure. Alternatively, higher pressures and/or temperatures could be used.

The plant extract can then be separated from the solids (i.e. the remaining plant material) by methods known to the skilled person, for example by filtration. Filtration using Whatman paper may be used.

If necessary, the plant extract can then be stored for future use, preferably at about 4 °C.

Step (ii)

In this step, a metal salt is added to the plant extract, which is preferably followed by a base.

If used, the base is preferably added dropwise over a period of time, such as from about 10 minutes to about 2 hours, preferably from about 30 minutes to about 90 minutes. Any base is preferably added whilst the mixture is heated, wherein the temperature is preferably from about 50 °C to about 100 °C, more preferably from about 70°C to about 97 °C and more preferably from about 85 °C to about 95 °C.

Any base is preferably added in sufficient quantity to cause the pH of the mixture to increase to from about 10 to about 12, such as from about 10.5 to about 11.5.

It is believed that controlling the pH allows for better precipitation of the metal oxide nanoparticles.

After the metal salt and plant extract have been contacted, and after any base has been added, the mixture is generally stirred for from about 1 to about 10 hours, preferably from about 3 to about 5 hours. During this time the mixture is generally maintained at the same temperature.

The metal salt is preferably a salt of zinc. Suitable zinc salts include zinc nitrate (preferably zinc nitrate hydrate, Zn(NOs)2-6H2O), and zinc sulfate. The metal salt may be in a concentration of from about 0.1 to about 1 M, such as from about 0.4 to about 0.6 M.

If used, the base is preferably a metal hydroxide, such as sodium hydroxide or potassium hydroxide, preferably potassium hydroxide. The base is preferably used in a concentration of about 1 to about 3M, such as about 1.5 to about 2.5 M.

The metal salt is generally added in the form of a solution, preferably an aqueous solution.

The progress of the reaction can be monitored by visual inspection, for example by monitoring for a colour change. In one embodiment the colour changes from colourless to a light-yellow colour. The colour change can also be monitored using UV-vis spectrometry.

The solution containing the nanoparticles can subsequently be separated in order to isolate the solid nanoparticles. This may be done passively (e.g. by allowing the solution to separate), and/or using active means such as filtration, for example using Whitman paper and/or a centrifuge.

If required, the nanoparticles may then be washed, for example using water and/or ethanol.

If required, extra steps can be added in the purification process, such as an intense vortex treatment of the precipitate along with the cleansing solution before each centrifugation plus brief ultrasonic exposure for better homogenization.

Finally, the nanoparticles may be dried or dehydrated. This step may involve heating the nanoparticles for a period of time.

The nanoparticles formed using the biosynthetic method described herein may then be used in the biofortification processes described herein.

Zinc oxide nanoparticles produced by the biofabrication method described above using Salvia fruticosa and/or Thymbra capitata(L.) Cav. generally have hexagonal crystals with a size of 22 +/- 4 nm. Thus, in one aspect the invention provides zinc oxide nanoparticles having hexagonal crystals with a size of 22 +/- 4 nm.

Examples Example 1 - Bio-fabrication

In the first step, the preparation of a plant extract rich in secondary metabolites takes place, after harvesting and drying of plant leaf blades. Drying incorporates air-drying at room temperature (25 °C) in dark conditions for about 10 days.

After harvesting and drying, 7 g dry weight of the plant material was purified/washed out with distilled water and was then pulverized with liquid nitrogen in a pestle and mortar until a powder was formed.

The ground material was added to 70 ml of double distilled water, and then magnetically stirred at 60 °C for 25 minutes before being allowed to cool down to room temperature. The material was then filtered with ashless Whatman paper N.40 (8 pm pore size) and the supernatant (plant extract) was stored at 4 °C for further treatments.

In the next step, 2M potassium hydroxide (KOH) and 0.5M zinc nitrate (Zn(NOs)2-6H2O) were prepared. The zinc nitrate solution was stirred for 30 minutes, then 10 ml of the plant extract was added, and the heating plate set at 90 °C. Afterwards, in a procedure lasting approximately 1 hour, the potassium hydroxide solution was drop-wisely infused until the pH reached 11. Stirring was continued for another 4 hours at 90 °C until the appearance of a light-yellow colour in the precipitate. Subsequently, the solution was incubated overnight at room temperature for a passive phase separation and was then centrifuged at 7000 rpm for 15 minutes for the isolation of the precipitate.

The precipitate was then washed with double distilled water and ethanol. If required, extra steps could be added in the purification process, such as an intense vortex treatment of the precipitate along with the cleansing solution before each centrifugation plus brief ultrasonic exposure for better homogenization.

Finally, dehydration of the pellet at 90 °C and calcination in the oven for 2 hours at 550 °C was carried out. This method resulted in the direct isolation of zinc nanoparticles without repeatedly stirring and heating.

Example 2 - Biofortification The concentration of zinc (in ppm) was measured in foliage samples of Salvia fruticosa plants after exposure to spray treatment with (i) distilled water, (ii) Zn(NOs)2 (2.1906 g/L in distilled water) and (iii) ZnO NPs (1 g/L in distilled water) produced according to the present invention.

The results are shown in Figure 1 , which show that the biofortification using the zinc oxide nanoparticles of the present invention was highly effective.

There are many alterations in the concentration of specific components of the essential oils after foliar spraying with Zn(NOs)2 or ZnO NPs. In particular, foliar spray with a Zn source leads to a percentage increase in the concentration of some substances (P-Pinene, Myrcene, p-Cymene, 1,8-Cineol, y- terpinene, Linalool, trans-Pinocamphone, a-Terpineol, 6- Terpinyl acetate, a-Ylangene, a-Copaene, p-Caryophyllene, y-Elemene, trans- -Farnesene, y-Muurolene, Alloaromadedrene) and to a percentage reduction of other substances (a- thujene, a-Pinene, Camphene, Limonene, Camphor, Borneol, Bornyl acetate).

The concentrations of components of particular interest (such as p-Cymene, 1,8-Cineol, a- terpineol, linalool, trans-thujone, camphor, p-caryophyllene, y- terpinene, borneol) in the treated S. fruticosa and/or S. fruticosa extract obtained after the above-mentioned treatments (i)-(iii) were also modified, as set out below. p-Cymene is a component of S. fruticosa essential oil with a content of 0.08% in the control sample (i.e. oil obtained from S. fruticosa treated with distilled water). Spraying S. fruticosa with Zn(NOs)2 before harvesting increased the content of p-cymene in the S. fruticosa extract by 381.25%, whilst spraying the S. fruticosa with ZnO NPs increased the content of p- cymene by 181.25%. Therefore, spraying S. fruticosa with zinc oxide nanoparticles significantly enhanced p-Cymene production.

The 1,8-cineol content in S. fruticosa essential oil was also found to increase after treatment with a Zn source. Specifically, spraying S. fruticosa with Zn(NOs)2 caused an increase in the amount of 1 ,8-cineol in the final plant extract from 41.04% to 41.36%, whilst spraying the S. fruticosa with ZnO NPs raised it to 44.53%.

Chemical analysis also showed that untreated S. fruticosa leaves had a 2.575% content of a-terpineol, and that treatment with Zn(NOs)2 as raised it to 4.91% whilst treatment with ZnO NPs raised it to 5.69%. Linalool is also a component of S. fruticosa essential oil. It is observed that the addition of a source of Zn enhances its production from 0.78% to 1.925% and from 0.78% to 0.95% by spraying with Zn(NOs)2 and ZnO NPs respectively.

Thujone is also a component of S. fruticosa-, 3,495% cis-thujone and 10,205% trans-thujone. The two diastereomeric forms show the opposite change by spraying with Zn(NOs)2 and ZnO NPs with the cis form almost doubling in the zinc nitrate spray samples whilst tripling in the ZnO NPs spray samples. Conversely, the trans form is tripled with Zn(NOs)2 spray and increased 1.4 times with ZnO NPs spray.

Camphor content decreased from 9.303% in the control sample to 5.855% after treatment Zn(NOs)2 and to 2.09% in samples with ZnO NPs.

P-caryophyllene is found to be component of S. fruticosa oil, and spraying with a Zn source achieved an increase of its production. Specifically, an increase from 1.915% (control) to 5.5% was observed after the Zn(NOs)2 spray and to 4.22% after the ZnO NPs spray. y-Terpinene is a component of Salvia fruticosa essential oil and its content also increased after zinc foliar spraying treatment, from 0.75 % (control) to 1.035 % and 0.85 % in Zn(NOs)2 and ZnO NPs treated plants respectively.

In the control samples of Salvia fruticosa essential oils, borneol comprises 1.12%, but shows a reduction in response to zinc foliar spraying of the plants. Specifically, after treatment with Zn(NOs)2 the borneol content fell to 0.475 % and by adding ZnO NPs it fells under a detectable limit.

Preferred embodiments:

1 . A composition comprising powdered plant material from a plant selected from the group consisting of Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof, and wherein the plant has been biofortified with a metal.

1a. The composition of any preceding embodiment, wherein the composition further comprises powdered plant material from a second plant which is different to the first plant.

1 b. The composition of embodiment 1 a, wherein the second plant is a plant of the families Lamiaceae or Rutaceae, Zingiber officinale or Aloysia citrodora. lc. The composition of any preceding embodiment, wherein the composition further comprises a compound providing a taste or health benefit. ld. The composition of embodiment 1c, wherein the compound providing a taste or health benefit is a vitamin.

2. The composition of any preceding embodiment, wherein the powdered plant material has an average particle size of between about 1 and about 100 pm.

3. The composition of any preceding embodiment, wherein the powdered plant material has an average particle size of between about 5 and about 50 pm.

4. The composition of any preceding embodiment, wherein the powdered plant material has an average particle size of between about 15 and about 20 pm.

5. The composition of any preceding embodiment, wherein the plant material comprises leaves, roots, rhizomes, bark, fruits, flowers, or combinations thereof.

6. The composition of any preceding embodiment, wherein the plant material comprises one or more leaves.

7. any preceding embodiment, wherein the plant is selected from the group consisting of Salvia fruticosa Mill., Aloysia cotriodora Palau, and combinations thereof. 8. The composition of any preceding embodiment, wherein the plant is Salvia fruticosa Mill.

9. The composition of any preceding embodiment, wherein the composition is a foodstuff.

10. The composition of any preceding embodiment, wherein the composition is in the form of macha tea.

11. The composition of any preceding embodiment, wherein the powdered plant material has a higher metal content than powdered plant material from an otherwise equivalent plant which has not been biofortified.

12. The composition of any preceding embodiment, wherein the powdered plant material has a higher p-cymene content than powdered plant material from an otherwise equivalent plant which has not been biofortified.

13. The composition of any preceding embodiment, wherein the powdered plant material has a higher 1,8-cineol content than powdered plant material from an otherwise equivalent plant which has not been biofortified.

14. The composition of any preceding embodiment, wherein the powdered plant material has a higher a-terpineol content than powdered plant material from an otherwise equivalent plant which has not been biofortified.

15. The composition of any preceding embodiment, wherein the powdered plant material has a higher linalool content than powdered plant material from an otherwise equivalent plant which has not been biofortified.

16. The composition of any preceding embodiment, wherein the powdered plant material has a higher trans-thujone content than powdered plant material from an otherwise equivalent plant which has not been biofortified.

17. The composition of any preceding embodiment, wherein the powdered plant material has a higher p-caryophyllene content than powdered plant material from an otherwise equivalent plant which has not been biofortified. 18. The composition of any preceding embodiment, wherein the powdered plant material has a higher y-terpinene content than powdered plant material from an otherwise equivalent plant which has not been biofortified.

19. The composition of any preceding embodiment, wherein the powdered plant material has a lower camphor content than powdered plant material from an otherwise equivalent plant which has not been biofortified.

20. The composition of any preceding embodiment, wherein the powdered plant material has a lower cis-thujone content than powdered plant material from an otherwise equivalent plant which has not been biofortified.

21. The composition of any preceding embodiment, wherein the powdered plant material has a lower borneol content than powdered plant material from an otherwise equivalent plant which has not been biofortified.

22. The composition of any preceding embodiment, wherein the metal is zinc.

23. The composition of any preceding embodiment, for use in treating or preventing metal deficiency or related disorders.

24. The composition of any preceding embodiment, for use in treating or preventing zinc deficiency.

25. A method of treating or preventing a metal deficiency or related disorder in a person, the method comprising orally administering the composition of any preceding embodiment.

26. A method of treating or preventing zinc deficiency in a person, the method comprising orally administering the composition of embodiment 21.

27. A method for forming the powdered plant material of any of embodiments 1- 21 , the method comprising

(a) fortifying a plant selected from the group consisting of Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof, with a metal; (b) harvesting plant material from the plant(s);

(c) optionally drying the plant material;

(d) forming the plant material into a powder (e.g. by grinding).

28. The method of embodiment 27, wherein step (a) comprises applying metal oxide nanoparticles to a plant selected from the group consisting of Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof.

29. The method of any of embodiments 27-28, wherein step (a) comprises applying metal oxide nanoparticles to the foliar of a plant selected from the group consisting of Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof.

30. The method of any of embodiments 27-29, wherein step (a) comprises spraying metal oxide nanoparticles onto the foliar of a plant selected from the group consisting of Salvia fruticosa Mill., Aloysia cotriodora Palau, Lavandula stoechas L., Lavandula angustifolia Mill., Melissa officinalis L., Ocimum basilicum L., or combinations thereof.

31. The method of any of embodiments 27-30, wherein the metal oxide nanoparticles are in the form of a solution comprising nanoparticles and a carrier or solvent

32. The method of embodiment 31 , wherein the carrier or solvent is water.

33. The method of any of embodiments 31-32, wherein the nanoparticles are present in the solution at a concentration of from about 0.1 g/L to about 5 g/L.

34. The method of any of embodiments 31-33, wherein the nanoparticles are present in the solution at a concentration of from about 0.5 g/L to about 2.5 g/L.

35. The method of any of embodiments 31-34, wherein the nanoparticles are present in the solution at a concentration of about 1 g/L. 36. The method of any of embodiments 27-36, wherein the metal oxide nanoparticles are zinc oxide nanoparticles.

37. A method of fortifying Salvia fruticosa Mill, and/or Aloysia cotriodora Palau, comprising applying metal oxide nanoparticles to Salvia fruticosa Mill, and/or Aloysia cotriodora Palau.

38. The method of embodiment 37, wherein the metal oxide nanoparticles are applied to the foliar of Salvia fruticosa Mill, and/or Aloysia cotriodora Palau.

39. The method of any of embodiments 37-38, wherein the metal oxide nanoparticles are applied by spraying the nanoparticles onto the Salvia fruticosa Mill, and/or Aloysia cotriodora Palau.

40. The method of any of embodiments 37-39, wherein the metal oxide nanoparticles are in the form of a solution comprising nanoparticles and a carrier or solvent

41. The method of any of embodiment 40, wherein the carrier or solvent is water.

42. The method of any of embodiments 40-41 , wherein the nanoparticles are present in the solution at a concentration of from about 0.1 g/L to about 5 g/L.

43. The method of any of embodiments 40-42, wherein the nanoparticles are present in the solution at a concentration of from about 0.5 g/L to about 2.5 g/L.

44. The method of any of embodiments 40-43, wherein the nanoparticles are present in the solution at a concentration of about 1 g/L.

45. The method of any of embodiments 37-44, wherein the metal oxide nanoparticles are zinc oxide nanoparticles.

46. The method of any of embodiments 37-45, wherein the metal oxide nanoparticles are applied to the foliar of Salvia fruticosa Mill.

47. A method for making metal oxide nanoparticles, the method comprising: (i) obtaining a plant extract from Salvia fruticosa Mill, and/or Thymbra capitata(L.) Cav.; and

(ii) adding a metal salt to the plant extract.

48. The method of embodiment 47, wherein the plant extract is formed from plant material selected from leaves, roots, rhizomes, bark, fruits, flowers, or combinations thereof.

49. The method of any of embodiments 47-48, wherein the method first comprises collecting or harvesting plant material from Salvia fruticosa Mill, and/or Thymbra capitata (L.) Cav., and then combining the plant material with water and heating to form an aqueous solution comprising the plant extract.

50. The method of embodiment 49, wherein the plant material is washed after collection or harvesting.

51. The method of embodiment 50, wherein the plant material is dried after washing.

52. The method of any of embodiments 49-51 , wherein the plant material is formed into a powder before being added to water.

53. The method of any of embodiments 49-52, wherein the heating step comprises heating a mixture of water and plant material to between about 30 °C and about 100 °C.

54. The method of any of embodiments 49-53, wherein the heating step comprises heating a mixture of water and plant material to between about 50 °C and about 70 °C.

55. The method of any of embodiments 49-54, wherein the heating step comprises heating a mixture of water and plant material for between about 10 and 60 minutes.

56. The method of any of embodiments 49-55, wherein the heating step comprises heating a mixture of water and plant material for between about 15 and 30 minutes. 57. The method of any of embodiments 49-56, wherein the heating step is carried out at atmospheric pressure.

58. The method of any of embodiments 47-57, wherein step (i) further comprises separating the plant extract from the remaining solids.

59. The method of any of embodiments 47-58, wherein a base is added after the metal salt.

60. The method of embodiment 59, wherein the base is added dropwise over a period of from about 10 minutes to about 2 hours.

61. The method of any of embodiments 59-60, wherein the base is added whilst the mixture is heated, wherein the temperature is from about 50 °C to about 100 °C.

62. The method of any of embodiments 59-61 , wherein the base is added whilst the mixture is heated, wherein the temperature is from about 70°C to about 97 °C.

63. The method of any of embodiments 59-62, wherein the base is added whilst the mixture is heated, wherein the temperature is from about 85 °C to about 95 °C.

64. The method of any of embodiments 59-63, wherein the base is added in sufficient quantity to cause the pH of the mixture to increase to about 10 to about 12.

65. The method of any of embodiments 59-64, wherein the base is added in sufficient quantity to cause the pH of the mixture to increase to about 10.5 to about 11.5.

66. The method of any of embodiments 59-65, wherein the method further comprises stirring the mixture for from about 1 to about 10 hours.

67. The method of any of embodiments 59-66, wherein the base is sodium hydroxide or potassium hydroxide.

68. The method of any of embodiments 59-67, wherein the base is used in a concentration of about 1 to about 3M. 69. The method of any of embodiments 47-68, wherein the metal salt is a salt of zinc.

70. The method of embodiment 69, wherein the zinc salt is zinc nitrate or zinc sulfate.

71. The method of any of embodiments 47-70, wherein the metal salt is present in a concentration of about 0.1-1 M.

72. The method of any of embodiments 47-71 , wherein the metal salt is added in the form of an aqueous solution.

73. The method of any of embodiments 47-72, wherein the resulting nanoparticles are dried or dehydrated.

74. The method of any of embodiments 47-73, wherein the resulting nanoparticles are zinc oxide nanoparticles having hexagonal crystals with a size of 22 +/- 4 nm.

75. Zinc oxide nanoparticles having hexagonal crystals with a size of 22 +/- 4 nm.

76. The method of any of embodiments 28-46, wherein the nanoparticles are formed using the method of any of embodiments 47-74.

77. The method of any of embodiments 28-46, wherein the nanoparticles are zinc oxide nanoparticles having hexagonal crystals with a size of 22 +/- 4 nm.