Plant extracts have been identified since ancient times. They have been used in the past, and are still used, in traditional Chinese medicine, Ayurveda and homeopathic medicine. Natural medicine is the oldest form of medical treatments. Examples of phytoactive ingredients are ginkgolides and flavone glycosides from Ginkgo biloba, bioflavonoids from citrus fruits and oleuropein/hydroxytyrosol from olives.
The choice is huge and to find the right extract for a specific application is not simple. One plant extract can have different activity profiles depending on the extract type and extraction method. The INCI declaration could be identical, even though the effect will be significantly different. Standardisation and defined production processes help to find the right products. Efficacy tests will definitely make the difference between having a marketing product with a nice label picture and an active ingredient with profound claim substantiation.
Herbal medicine is an aspect of indigenous medicine – the use of gathered plant parts to make teas, poultices or powders that purportedly effect cures. Relevant history should be considered.1 Indigenous medicine is sometimes associated with quackery when practiced as theatrics or otherwise practiced fraudulently, and sometimes with witchcraft and often with shamanism, yet it may also preserve important knowledge and cultural tradition from the past. The term traditional medicine (indigenous medicine or folk medicine) describes medical knowledge systems which developed over centuries within various societies before the era of modern medicine. Traditional medicines include practices such as herbal medicine, Ayurvedic medicine, traditional Chinese medicine, as well as other medical knowledge and practices all over the globe. Early recognised compilers of existing and current herbal knowledge were the Greeks Hippocrates, Aristotle, and Theophrastus. Roman writers were Pliny and Celsus. Early Greek and Roman compilations became the backbone information sources of European medical theory and were translated by Arab, Persian and Jewish scholars.
Examples of phytoactive plant ingredients Ginkgolides/flavone glycosides The Ginkgo tree is the only still existing representative of the genus Ginkgo plants that had their main development around the time of dinosaurs. As the plant has remained unchanged up to today it is called a “living fossil”. In contrast to Western culture, where mainly the leaves were used, the seeds came to the fore in traditional Chinese medicine. The Ginkgo seeds were a valued drug against tuberculosis which is substantiated with their antibacterial properties. Further application areas of the seeds (baigo) include those for asthma, cough and cystitis. The first secured notations originate in the year 1436, when the topical application of Ginkgo leaves to counter ski diseases and inflammation, and to act as a first aid measure, was described.2 Ginkgolides have anti-inflammatory properties. Terpene lactone ginkgolides are unique and can be found in Ginkgo biloba trees only. They are diterpenes that are slightly different in structure. The terpene lactone bilobalide has a sesquiterpenoide structure. The effects of Ginkgo flavone glycosides derive from anti-oxidative properties, specifically from the possibility to act as a radical scavenger against oxygen-free radicals. The accumulation of oxygen-free radicals is a particularly large factor in age-related change processes in skin. Ginkgo flavone glycosides protect against peroxidation of lipids that are important components of the cell membranes and essential for the preservation of the membrane flexibility. The radical scavenger function of Ginkgo biloba is comparable with other well-known antioxidants such as vitamin C and E.3
Oleuropein/hydroxytyrosol Anti-oxidative properties of the polyphenols from olive oil and leaves can be shown in various tests. Le Tutour et al found olive leaf extract, oleuropein and hydroxytyrosol to be stronger in anti-oxidative power than vitamin E and BHT. The extract showed the best effect due to the high content of oleuropein and due to the lower content of flavonoids. Visioli et al investigated the anti-oxidative properties of oleuropein and its metabolites in a series of experiments. Excellent radical scavenger properties and inhibition of copper sulphate-induced LDL oxidation were found.4 Depending on the olive’s degree of ripeness, oleuropein and hydroxytyrosol are found in the cold pressed oil (approx. 0.1%) and develop effects similar to omega-3 fatty acids, especially the oleuropein. With increasing maturity of the olive, hydroxytyrosol will be released, which is stronger in anti-oxidative power. The amphiphilic hydroxytyrosol is a very strong antioxidant (about 100 times stronger than vitamin C according to a DPPH-test).6 As hydroxytyrosol is soluble in fat as well as in water, it can develop its effect as a powerful radical scavenger in cell membranes as well as in the cell plasma.5 Hydroxytyrosol protects in vitro human melanocytes from protein damage induced by long-wave UV light, and reduces the release of inflammation inhibitors such as Cox-2 in macrophages.6,7.
Citrus bioflavonoids: hesperidin, naringenin, eriodictyol Bioflavonoids are oil soluble and high concentrations can be found in cell membranes after oral uptake. They are strong antioxidants, neutralising superoxide and hydroxyl radicals. Hesperetin, particularly the aglycone hesperidin, and naringenin protect the DNA and induce the formation of phase II enzymes that provide detoxifying properties to toxins and chemicals.8 Phase I Enzymes are involved in the modification of chemical substances which are needed by the body, adding a simple nitrogen or oxygen molecule. They are also called “Activators”. This very small change facilitates the move to the next phase. Phase II Enzymes are called “Excreters” and take over from the Phase I Enzymes. They bind the modified molecules to another small molecule namely glutathione. This connection enables the body to excrete substances because they were made water soluble.
Choice of suitable extracts
The number of extract suppliers is huge. The INCI denomination of extracts could be identical, even though the ingredients and their concentrations could be totally different.
Natural extracts In natural extracts the concentration of actives is dependent on the concentration in the plant. The solvent (particularly the the solvent mixture) has a major influence. The solubility of the plant active in a solvent or solvent mixture and the extraction process, whether maceration or percolation, have important roles. Standardised extracts offer higher active contents. Standardisation means the adjustment of a defined content of a substance or a substance group which determines efficacy (according to “Normieren” nach F.W. Hefendehl und C. Lander [BfArM], 1982).
Standardisation concept A drug is characterised through comprehensive specifications such as cultivation, origin, harvest, drying, and storage. The solvent is characterised by its type and concentration. The production process must be optimised and validated regarding method of extraction, temperature, time, etc. The residual solvent, the active ingredient as the “marker”, and the microbiological status, have to fulfil given specifications. A standardised extract is characterised by its production process, the specification, the validation and stability data.9 The quality of the plant active is determined by the process. The plant active is characterised by the kind of its efficacy tests which might be in vitro, ex vivo or in vivo and the chemical analysis.
Screening tests
In vitro screening tests
The efficacy of plant actives can be determined by a number of different tests. In vitro tests are based on grown skin cells that are inoculated with the diluted plant active. A huge number of methods are available. Different skin cells, growth media, detection substances and detection methods can be chosen according to the specific question of the investigator. The biochemical pathway through the skin can be determined and results provided for certain claim substantiations. Conclusions for supplementary in vivo tests are possible. Practically, two different main skin models are used: 2D and 3D models. A 2D model is a simple cell culture. The actives are added to the cell medium. The concentration relationships are totally different to a real skin application. There is no barrier structure such as the stratum corneum which is always present in human skin. Only water-soluble ingredients can be tested, however, complex cream formulations cannot be tested. Only one cell type per test can be evaluated, e.g. keratinocytes, fibroblasts, melanocytes. The correlation between the stratum corneum, the epidermis and the dermis cannot be taken into account.10 The 3D model opens new ways for in vitro skin tests. By cultivating a dermis from fibroblasts and an epidermis with a stratum corneum from keratinocytes, a “whole skin model” develops within a time frame of 5 weeks, containing all essential layers of the skin. After cultivation the “whole skin model” lives for about 4 further weeks almost unchanged. During this time anti-ageing experiments such as UV irradiation or ozone treatment can be applied, and also topical applications for skin protection or skin care can be used. The major advantage is that these skin models have a stratum corneum like normal human skin. The actives can be applied under realistic conditions in a cream or gel form. The actives penetrate the stratum corneum and deploy their efficacy.10
Ex vivo screening tests Excised human skin can be taken for ex vivo studies. The use of so-called diffusion cells for ex vivo studies is based on the thesis that the barrier properties of excised skin are identical with in vivo conditions. The thesis could be confirmed by many investigations. Ex vivo studies assess the skin penetration and metabolism of topically applied actives in human skin. For a study fresh surgically excised human abdominal skin is mounted on Franz perfusion cells. A defined quantity of the test substance is applied on the surface of the mounted skin. Measurements can be made with cosmetic formulations and diluted actives. Conclusions about the penetration depth and the corresponding time gradient of the active through the skin can be drawn. Claim substantiation is possible.
In vivo tests
Studies are based on data from recruited human volunteers. The final cosmetic formulations are applied on the skin and their efficacy can be tested with instrumental measurements, dermatologist readings and/or questionnaires. The test result can be measured directly which allows the substantiation of strong claims. The applied formulation has a strong influence on the efficacy of cosmetic actives.11 The conclusion of the test can differ between different formulations. In vivo tests do not give hints about the biochemical pathway of the actives in skin.
In vitro screening test examples Cytotoxicity Before an in vitro test can be performed, the cytotoxicity of the test substance has to be determined. An example for a cytotoxicity test is the Quantitative Growth Inhibition Test (MTT Test). The evidence of cell viability with the MTT test is based on the reduction of a yellow, water-soluble colorant (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazoliumbromid =MTT) into a blue violet, water-insoluble formazan. The colour change only happens when mitochondria in the cells are viable. The results give a direct indication about the ideal use concentration of the active for in vitro trials. The highest concentration that is about to reach 100% cell viability should be chosen. ATP reduction after UV irradiation Biochemical cell processes need energy. ATP (Adenosine triphosphate) is the energy reservoir in the cells and is involved in a number of different tasks. ATP is needed for cellular movements and the movement of chromosomes during mitosis. It is also necessary for the transport of molecules against a concentration gradient. The synthesis of cellular macro-molecules such as DNA, RNA, proteins and polysaccharides is supported. The higher the ATP quantity remains after induced cell damage, the better the cell can activate its own protection shield.
ATP level in keratinocytes after UV irradiation A cell culture test to measure the amount of ATP can be performed on epidermal keratinocytes. Cultures of keratinocytes with and without an active ingredient are grown under the same conditions in a full medium. They are irradiated with a defined dose of UV light, and the measurement of the remaining ATP quantity of both cultures is taken. If the active ingredient has a positive influence on the keratinocytes, the ATP quantity is higher in comparison with the untreated control.
DNA repair
DNA damage is one of the most acute threats to cellular homeostasis and life. The cell responds to such damage by activating a number of responses, ranging from DNA repair to numerous signalling pathways temporarily slowing down the cellular life cycle while the damage is being repaired. Sophisticated relays convey the DNA damage alarm to all these systems immediately after damage infliction. Such relays must be capable of sensing the damage and rapidly creating functional contact with many signalling networks.12 Genome replication is a delicate and complex process which is prone to alterations in DNA sequences. The greatest threat to genome stability is the damage caused by physical and chemical agents. Damaging chemical agents are even formed during normal cellular metabolism, e.g. reactive oxygen species (ROS), or may come from the environment. They range from free radicals to compounds that react with DNA components to alter DNA constituents or induce strand breaks. An example of a single strand damage and a built-in repair mechanism is the formation of a dimer after UV irradiation. Two thymine nucleotides can dimerise. The damaged part of the strand will be excised and hydrolysed. A repaired nucleotide chain will be integrated into the double DNA strand (Fig. 1). A particularly toxic lesion is the doublestrand break (DSB). Even one unrepaired DSB is lethal to the cell (Fig. 2). DNA damage triggers processes in the cell that lead either to damage repair and safe resumption of the cellular life cycle, or to programmed cell death, the so-called apoptosis. The scope of cellular responses to DNA damage is considerably larger than the processing and removal of the DNA lesion, particularly when it comes to critical types of DNA damage such as DSB. A prominent cellular response to such damage is the activation of the cell cycle checkpoints that temporarily arrests the cell cycle. The damage response thus involves the concomitant modulation of an intricate network of pathways. The apoptosis is a structured, ATP depending process induced by external and internal signals. After induction it passes phases which are determined by genetically fixed information. The cells undergo morphological changes during apoptosis.
Stimulation of DNA repair after UV irradiation
The DNA of human keratinocytes, cultivated with and without active ingredient can be damaged with UV irradiation. Two examples for the detection of the active ingredients efficacy are:
• BrdU incorporation.
• Nucleosome formation.
• Fragments of the cell nucleus.
BrdU incorporation BrdU (Bromodesoxyuridine) is homologeous to thymine and will be offered during the repair occurence. BrdU will be incorporated instead of thymine, when it is offered in a higher concentration. It can be detected with the help of an anti-BrdU antibody. The incorporation rate of BrdU instead of thymine reflects the repair efficacy of the cells.
Nucleosome formation Nucleosomes are nucleus fragments that are formed when apoptosis starts. In keratinocyte cultures nucleosomes are formed after irradiation. Many fewer nucleosomes are formed when an active ingredient protects the cells against DNA damage in comparison to an untreated control.
Oxidative stress Aggressive chemical components, so-called free radicals, are formed in an organism under the influence of oxidative stress. Reactive oxygen species (ROS) are mainly developed through the respiratory chain (Fig. 5) inside the human body. How can oxidative stress occur in our body? With every breath we take up half a litre of air, consisting of 20% oxygen. All living cells in our body need energy to work, and thus oxygen. In the mitochondria, the power stations of our body, energy is produced in series of reactions with oxygen. As the power stations are not perfect, 1% to 2% of the oxygen used in the mitochondria is reduced by only one electron. As a result, the superoxide radical O2 –• is formed, able to form hydrogen peroxide, H2O2. Neither superoxide nor hydrogen peroxide alone are particularly dangerous. But the reaction of the two, especially in presence of heavy metal ions such as copper or iron, can release the extremely reactive hydroxyl radical OH•, thus seriously damaging the cell (Fig. 7).13 To investigate oxidative stress, flow cytometry can be used.4 For the detection of a reaction on human keratinocytes DHR (dihydroxyrhodamine) was added. Irradiation with UVB light induced the release of H2O2. DHR is transformed into the fluorescent rhodamine (Fig. 8).
Benefit and use of the results
ATP content
If the ATP content remains high in damaged cells their own repair mechanism will function well. Cell functions remain totally intact and the defence forces are high.14
Claim propositions w Strong own body repair.
• Strong power of resistance.
• Strengthening of the skin’s own protective function.
• Strengthening of the skin’s defensive forces.
• Keeps the skin healthy.
DNA repair
The DNA repair rate can be improved when cosmetic actives are used. The increase of the repair rate demonstrates efficacy of the DNA protection. This can be seen by a low rate of double strand breaks and the number of cells that must be destroyed by the body via apoptosis remains small. Mitochondria are the only cell organelles with their own DNA. Opposite to the nucleus DNA, the mitochondria DNA cannot be renewed. In case of damage occurrences the performance of the cell decreases. Avoiding damage keeps the cells at a high performance level for longer.14
Claim propositions
• Improves mature skin’s DNA repair capacity.
• Anti-ageing properties. w Strengthening the skin’s cell structure.
• Active cell protection.
Oxidative stress
The formation of free radicals in membranes and cell organelles, e.g. mitochondria, is more often a problem of mature skin in comparison to juvenile skin. However, oxidation processes regularly happen because of internal and external influences. The problems are often recognised in cell membranes and the extracellular matrix. The formation of free radicals is not limited to mature skin only. Long and intensive sun exposure or longlasting stress situations will also have an influence on juvenile skin.
Claim propositions
• Against free radicals.
• Prevents premature skin ageing.
• Protects skin against environmental influences.
• Anti-ageing. w Keeps skin resilience.
• Strengthens defence forces.
Conclusion
Plant extracts are a manifold source for cosmetic actives. Prepared as a standardised product with defined marker substances they can have substantial efficacy. The way such an active ingredient will be supported is important for the success. The understanding of the biochemical pathway of an active ingredient is becoming more important as it offers new claim ideas. The substantiation of one specific claim, however, needs more than one test and the tests should be complementary. An ATP level could be combined with the demonstration of an increased collagen level after a treatment. Both tests together could be a promising basis for an antiageing claim. DNA repair and oxidative stress could lead to a claim of protection against environmental influences or against premature skin ageing. Combinations of tests reinforce an interesting and strong marketing story. An in vitro test for background information of the biochemical pathway of an active ingredient cannot replace an in vivo test for the final formulation. In vitro tests do not support claims against wrinkle depth or skin elasticity or moisturisation, in vivo tests do so. Both sorts of tests support a cosmetic product. The complementary combination will strengthen a single claim, thus giving a more comprehensive picture of the active ingredient.
References
1 Traditional medicine from Wikipedia, the free encyclopedia.
2 Ginkgo biloba. Susanne Krell. Naturheilpraxis 05/2002 www.naturheilpraxis.de
3 Ginkgolides, diterpene trilactones of Ginkgo biloba, as antagonists at recombinant a1h2g2L GABAA receptors. Shelley H. Huang, Rujee K. Duke, Mary Chebib, Keiko Sasaki, Keiji Wada, Graham A.R. Johnston. European Journal of Pharmacology 494 (2004) 131– 138.
4 Hydroxytyrosol from Olives, an interesting radical scavenger for cosmetic applications. Maria Lueder, Joachim Blank. CSC-Conference, Amsterdam, April 2008.
5 Informationsgemeinschaft Olivenöl (München): Wissenschaftliche Erkenntnisse über Olivenöl in der Ernährung. Hintergrundinformation Nr. 1 des Instituts für Arteriosklerose Forschung an der Westfälischen-Universität, Münster (November 2001).
6 Hydroxytyrosol, a natural antioxidant from olive oil, prevents protein damage induced by longwave ultraviolet radiation in melanoma cells. Free Radical Biology and Medicine 1; 38 (7): 908-19. April 2005. Stefania D’Angelo et al.
7 Hydroxytyrosol, a phenolic compound from virgin olive oil, prevents macrophage activation. Naunyn-Schmiedeberg’s Archives of Pharmacology. 2005 June. 371 (6): 457-65; Maria Chiara Maiuri et al.
8 DocMedicus Vitalstofflexikon. www.vitalstofflexikon. de
9 Definition und Klassifizierung pflanzlicher Drogenzubereitungen und daraus abgeleitete Probleme. Frauke Gaedcke. Forum Pharmaceuticum Okt. 2005.
10 Systematische Bewertung neuer Wirkstoffe und Kosmetika Förster Th., Jassoy C., Petersohn D., Schlotmann K., Waldmann-Laue M. Dermo Topics. März 2001.
11 Formulating for efficacy. Wiechers J.W., Kelly C.L., Blease T.G., Dederen J.C. International Journal of Cosmetic Science. Volume 26 Issue 4, Pages 173–182, July 2004.
12 ATM (ataxia telangiectasia mutated): expanding roles in the DNA damage response and cellular homeostasis. Shiloh Y. The David and Inez Myers Laboratory for Genetic Research, Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
13 Geistig fit durch mediterrane Kost; Gunter P. Eckert, Sebastian Schaffer, Stephanie Schmitt- Schillig, Walter E. Müller. Forschung Frankfurt 1/2003.
14 An anti-aging concept plus protection. Lueder M., Petersen R.D., John S., Borchert S. C&T Manufacture Worldwide. April 2003.
