Identification, Authentication and Extraction of Herbs

Herbs: The word herbs derived from the Latin word herba,which means grass, green stalks, or blades.
Herbs include any parts of the plant like leaves, roots, flowers, seeds, root bark, resin and pericar

Medicine is a substance that has nutritive, curative, or preventive properties, while the term “herbal” refers to a botanical or plant-based preparation. Hence, the term “herbal medicine” is used for plant-based substances that consist of nutritive, curative, or preventive properties. Herbal medicine is an interdisciplinary branch between herbal medicine and Ayurveda as it covers all fields of herbal medicine related to botany, medicinal plant research, pharmacognosy, phytochemistry, phytotherapy, botanical medicines, Ayurveda, natural chemistry, agriculture science, Unani medicine, biotechnology, and biochemistry. A person who deals with herbs, especially medicinal herbs, is known as an herbalist. Herbal journals deal with the use of plants in the treatment of diseases HM includes preparations of biologically active natural products that consist largely of herbs or herbal materials, some recipes may contain materials such as fungal and bee products, as well as minerals (kaolin, bentonite), ash, shells, insects and animal parts, and are used for the maintenance of health and management of various diseases.

HMs can elicit numerous benefits just as some can cause adverse effects. The pharmacologic and most of the toxic effects that are elicited by HMs have been linked to the activities of the secondary metabolites. In many instances, HMs have been appropriately used, misused and sometimes misunderstood. The benefits of HMs as a means of healthcare depends largely on the correct and adequate knowledge, and experiences while misuse as well as misunderstanding have been tracked to the knowledge gap on herbal medicines especially as it relates to their benefits and potential drawbacks by the primary healthcare professionals: doctors, pharmacists, nurses and the public. The attraction to herbal medicine will continue to increase across the globe for various reasons, hence the urgent need for appropriate and enough information on HM especially that which highlights on important topics such as benefits, efficacy, safety, toxicity, research and development, formulation, regulation, analytical techniques, quality control, economic importance, and so on. This book harnesses important information on various aspects of HM, thus, serving as a compendium to enlighten scientists, healthcare professionals and lay users appropriately.

With many people now using herbal medicine, safety issues are also becoming an important concern. Indeed, certain HM have been implicated in some important adverse events relating to cardio-, neuro- and nephro-toxicities as well cancers. Toxicity due to HMs may occur and their seriousness may vary depending on the type of herb or herbal material, preparation and user: varying from minor to severe and sometimes fatal. Adulterations and concomitant use of herbal medicines with conventional medicines constitute another area of attention, thus, the need for a strict regulation and enlightenment and control.

Different methods of Identification of plants:

Characters Considered Before Plant Identification:
i. Whether a plant is herbaceous or woody, and annual or perennial in nature.
ii. Whether or not milky or coloured sap is present in the leaf, stem or other plant parts.
iii. The leaf type, phyllotaxy, and venation.
iv. Presence or absence and type of stipule on young shoots.
v. The distribution and kinds of surface coverings (i.e. hairs, trichomes, spines, etc.).
vi. The parts of the flower and the number of sepals and petals, whether separate or fused, and also their arrangement i.e. aestivation.
vii. Whether perianth is present in one or more series, or absent.
viii. Whether pappus (e.g. Asteraceae) or epicalyx (e.g. Malvaceae) or similar structures are present.
ix. Whether a nectar-secreting disc is present in the flowers (e.g. Rutaceae).
x. Whether the flowers are actinomorphic or zygomorphic.

xi. The number and attachment of stamens and if there is any fusion of anthers or filaments.
xii. The number of pistils, styles and stigmas of the gynoecium, observation of a transverse section of the ovary, the number of locules, number of ovules per locule, and also the placentation.
xiii. The position of the ovary and fusion of the perianth by observing a longitudinally cut section of the entire flower through its centre.

Plant Identification Methods:

The methods of identification include the following:
(a) Expert Determination:
The best method of identification is expert determination in terms of reliability or accuracy. In general the experts have prepared treatments (monographs, revisions, synopses) of the group in question, and it is probable that the more recent floras or manuals include the expert’s concepts of taxa.
Experts are typically found in botanical gardens, herbaria, museums, colleges, universities, etc. However, although of great reliability, this method presents problems of requiring the valuable time of experts and creating delays for identification.

(b) Recognition:
It approaches expert determination in reliability. This is based on extensive, past experience of the identifier with the plant group in question. In some groups this is virtually impossible.

(c) Comparison:
A third method is by comparison of an unknown with named specimens, photographs, illustrations or descriptions. Although this is a reliable method, it may be very time consuming or virtually impossible due to the lack of suitable materials for comparison. The reliability is, of course, dependent on the accuracy and authenticity of the specimens, illustrations, or descriptions used in the comparison.

(d) The Use of Keys and Similar Devices (Synopses, Outlines, etc.):
This is by far the most widely used method and does not require the time, materials, or experience involved in comparison and recognition.

Authentication of plant:

Herb authentication is a quality assurance process that ensures the correct plant species and plant parts are used as raw materials for herbal medicines. The proper authentication of herbal raw materials is critically important to the safety and efficacy of herbal medicines.

Different extraction methods including advanced extraction techniques : supercritical fluid extraction

Supercritical fluid extraction is a novel technique especially used for the recovery of essential oil from plants. SFE is based on the use of carbon dioxide in supercritical phase, which is at low pressure and temperature (74 bar and 32°C): in this state CO2 possesses a polarity similar to pentane and so it is a good candidate for the extraction of lipophilic compounds. Furthermore supercritical CO2 is non toxic, non-flammable, not expensive, and easy to remove in the end of the process (eco-friendly). Operating at low temperature it is possible to obtain in high yield thermolabile compounds like terpenes and terpenoids that normally have their boiling point over 150°C and so it is important to work at lower temperatures for preventing their degradation. If the components to extract are polar, a cosolvent like water or ethanol in little percentage (5-10%) is needed to increase the extraction quality. When in the plant matrix there are bioactive compounds of different solubility, a method to improve the recovery of all the phytotherapeutics without any loss is the fractionation of the extract. Two strategies could be applied: the multi-step fractionation and the on-line fractionation.

When the multi-step fractionation is performed, there are different successive steps of separation with different conditions in terms of density of CO2: in the first step we will obtain the fraction of the more soluble compounds such as essential oils, whereas in the second step, increasing CO2 density we will have the recovery of the less soluble components like antioxidants. Moreover, thanks to this strategy we can obtain in the first step the products extracted by the only use of supercritical CO2, while in a second time we can add a cosolvent such as ethanol for the other compounds. The on-line fractionation works, instead, in a cascade depressurization system in order to obtain the precipitation of the several fractions according to their saturation conditions. The particular properties of the supercritical fluid characterized by a low viscosity and a high diffusion make this technique an excellent alternative to the others for the recovery of therapeutic products from plants.

Microwave assisted extraction

Microwave-assisted extraction (MAE) is an extractive method based on the utilization of microwave energy that is produced when the perpendicular oscillation between the electric and the magnetic fields generates electromagnetic radiations with a frequency ranging from 0.3 to 300 GHz. If the microwave goes through and interacts with a substance there is a production of heat whose intensity depends on the absorption of the energy by the material and the dissipation of the resulting heat.

MAE techniques can be classified according to the pressure through which they operate: higher than the atmospheric pressure (closed MAE system) and lower than the atmospheric pressure (open MAE system). As regards closed systems, the temperature is set over the boiling point of the solvent and the pressure is under control to avoid an excessive development. Among the closed systems we can enumerate high pressure microwave-assisted extraction (HPMAE) which uses high pressure and temperature in order to enhance the capacity of the solvent to incorporate the energy from radiation and to avoid large amount of solvent for the extraction. In case of thermolabile molecules, soft conditions are needed and so the choice will fall on an open system or the vacuum microwave assisted extraction (VMAE) that allows the reduction of the boiling point of the solvent. For compounds that are susceptible of oxidation it has been developed the nitrogen-protected microwave-assisted extraction operating under pressurized inert gas. When the bioactive compounds are susceptible of hydrolysis such as the essential oils, solvent-free microwave-assisted extraction (SFME) is used to avoid the loss/degradation of these products. Moreover, MAE can be associated to ultrasonic energy (ultrasonic/microwave-assisted extraction UMAE) to reduce extraction times and the amounts of solvent leading to an improvement of the yield. Another way to gain time is coupling the extraction step with the analytical one in the dynamic microwave-assisted extraction (DMAE) which operates continuously and automatically. The choice of the solvent is influenced by its ability to absorb the microwave radiation: ethanol, methanol, water and the more selective room temperature ionic liquids are good solvents for MAE. The ratio of solvent to solid is important for improving MAE: if the amount of solvent is too much it absorbs all the energy with a resulting inefficient matrix heating, whereas if the ratio is too low the amount of solvent is not enough to allow the diffusion of the compounds out of the matrix. Also the vessel size is a critical factor because in a little vessel the internal pressure tends to augment and this could mean a degradation of the more delicate molecules.

Another parameter to consider in MAE is the power of extraction: an increased power boosts the temperature reducing the solvent viscosity and leading to a better efficiency, except in case of thermolabile molecules. Microwave-assisted extraction gives several advantages with respect to classical extractive processes such as Soxhlet: MAE allows a gain of time, higher quality and yields. It is also cheaper than supercritical fluid extraction (SFE) and faster than ultrasonic-assisted extraction (UAE). On the other hand, MAE shows some drawbacks: it is more expensive than UAE, less eco-friendly than SFE due to the use of organic solvents, not suitable for thermolabile compounds because the irradiation could promote chemical reactions with the loss of the desirable products, and not efficient when the target molecules and/or the solvent of extraction are non-polar because they do not absorb energy from the source. This technique has never been applied to S. repens extraction.

Ultrasound assisted extraction

In recent times, ultrasound-assisted extraction has received a great interest to overcome the disadvantages of classical solvent extractions such as little yields and waste of time. UAE is based on the production of ultrasound waves and their transmission throughout the solvent with a resulting cavitation. When the cavitation bubbles collapse, there is a generation of liquid circulation currents and turbulence that improve the mass transfer rate. The fractures formed in the cell wall enhance its permeability and so a bigger amount of solvent can enter into the plant tissues to extract the bioactive metabolites. In order to perform an extraction based on sonochemistry, the choice of solvent becomes an important parameter because its physical properties like polarity, viscosity, vapour pressure and surface tension influence the cavitation phenomena. Ethanol, methanol and hexane are very used in UAE, and sometimes water could be added to ethanol, even if its amount must not be too much in order to avoid a decrease in extraction efficiency, probably due to the generation of radicals from the ultrasonic dissociation of water. Other parameters to be considered are the frequency and the power: often the former ranges from 20 to 100 kHz and the latter from 100 to 800 W. Also the power dissipation is a critical factor, because the generation of physical effects like turbulence is directly proportioned to the power dissipated as heat. For the future, the design of reactors based on multiple transducers is needed in order to operate at multiple frequencies and improve the efficacy of UAE. Another problem that currently limits the use of UAE at large scales is the erosion of transducers and their continuous replacement to avoid a decrease in the transmitted energy. Nonetheless, UAE is less expensive than the traditional extractive techniques; it can give high quantities of products without spending time and without using large amounts of solvent. For a better performance, UAE can be also used in combination with other techniques like supercritical fluid process. This technique has never been applied to S. repens extraction.

Isolation and purification techniques

  1. General Isolation techniques
  2. B. Extraction methods Plant material extraction is a crucial process in the isolation of natural plant compounds and their purification. Plant matrices naturally are complex, containing a wide range of compounds that have various physical and chemical properties. It is therefore imperative to carefully, isolate from the rest of the plant, matrices and make pure, compounds of interest in plants for their characterization. There are several ways extraction methods can be categorized. In this chapter, they have been categorized based on the temperatures they work under.

2.1 Low or room Temperature methods 2.1.1 Cold extraction method

The method has been described in literature. Briefly, dried plant parts samples (Cut, crushed or milled) are put in various solvents for seven days, with shaking every 24 hours. The samples are then filtered using a Whatman filter paper and dried under vacuum at room temperature on pre-weighed watch glasses and the mass of yield determined by difference. One common example of cold extraction is maceration. In this method, coarsely powdered plant part or whole plant material is stored in contact with a solvent for some time with

2.1.2 Enzyme Assisted Extraction (EAE)

This method employs solvents with various enzymes selected depending on the environments they perform the best and the pathway in mind that the scientist needs the compounds to be catalyzed. Some of the enzymes generally used in the extraction are protease, lipase and phospholipase and they effectively reduce the use of solvents. Specifically for essential oils, pectinase and α-amylase are the mostly used enzymes. The method is non-degrading to compounds but the setup is costly. It is also too demanding in terms of nutrients required, oxygen presence and temperature optimization

2.1.3 Plant tissue homogenization

Fresh, wet or dried parts of plants are ground and soaked in solvents. The mixture is either vigorously shaken for 5 to 10 minutes or let stand for 24 hours with regular shaking and then the extract is filtered. The extract filtrates may be centrifuged for clarified concentration then dried. Sometimes the filtrates may be directly dried under reduced pressure and then re-dissolved using solvents of interest. Most times, this requires a laboratory set up, and cannot be easily done in the field.

2.1.4 Ionic Liquid Extraction

In this method, organic salts in liquid form interact selectively with polar and nonpolar compounds using π- stacking interactions, hydrogen bonding, ion exchange and hydrophobic interactions. It is a very good method that recovers organic and inorganic ligands in high yields. Due to the ionic interactions, the quality and efficiency of the extraction is very high

2.2 High temperature extraction methods

Extraction in high temperatures should be conducted on compounds that are known to be thermally stable. Although extraction under elevated temperatures is feared by many to be denaturing to essential compounds, it is not all compounds that get destroyed. A research done on extraction of Asiatica cantella at understand the nature of the compounds of interest in the plant materials.

2.2.1 Decoction

This method involves boiling in water for 15 minutes, cooling, straining and passing appropriate amounts of cold water into the drug to give required volumes. Heat stable and water soluble compounds of interest from herbal plants can be extracted using this method .This method yields more oil-soluble compounds than infusion and maceration due to the high temperatures. This method is cheap and can be used in a field

2.2.2 Soxhlet extraction

This is best suitable when the compounds of interest are known to have limited solubility in a solvent and when impurities in that solvent are insoluble. Thermolabile compounds are not passed through this method as it can degrade them. The advantage of this method is that it does not require multiple passes of hot solvents, just one pass is enough as the same gets recycled. It requires a laboratory set up and Soxhlet apparatus.

2.2.3 Microwave assisted extraction

This is basically the traditional solvent extraction but the solvent and sample mixture is heated using microwave energy. The microwave energy penetrates through plant materials targeting and evaporating the smallest traces of moisture in plant cells. The cell walls rapture due to pressure created by the heating inside the cells. This rapturing exudates active compounds from within the cells, making this method more yielding of phyto-constituents.

2.2.4 Automated solvent digester extraction method

Recent use of universal extraction systems (BUCHI systems for example) employs thimbles which contain the test samples suspended in small glass jars containing various extraction solvents and temperature is set just below the solvent’s boiling point. The extracted extract is filtered and concentrated using vacuum concentrator, ready for phytochemical determinations.

2.2.5 Pressurized Liquid Extraction (PLE)

This employs an apparatus set up that uses heat of upto 200◦C and pressure of between 35 and 200 Bars. Samples are put in a sample holder, usually with water as a solvent. When the temperature is raised, the heat reduces viscosity of the solvent, making it easily penetrate the plant matrices. High pressure keeps the solvent in the liquid phase. The makeup of the apparatus protects oxygen and photo sensitive compounds from degradation. The method is ecofriendly but expensive. It is selectively good for thermally sable compounds.

2.3 Optional temperature extraction

The following methods can be undertaken optionally, depending on the knowledge of the nature of the constituents of interest in the samples.

2.3.1 Serial exhaustive extraction

This is extraction in a series of solvents from the least polar (usually n-hexane) to the most polar (Usually methanol) to ensure extraction of compounds at a wider range of polarities. It is one of the most common methods of extraction which can go either under elevated temperatures (eg. Soxhlet) or normal temperatures, particularly for thermolabile compounds.

2.3.2 Infusion and digestion

This method is used only when there are readily available components of the drug in crude form, like tea in tea bags. Infusions are freshly prepared by maceration of the ready drug components in hot or cold water. A closely related method is digestion, where during the preparation of the extract, a steady gentle heat is applied to the maceration process to enhance the release of active components.

2.3.3 Supercritical fluid extraction method

This method involves use of liquefied gases, usually CO2 which is pumped through a cylindrical channel The mixture is then taken to a separating chamber where the gases are recovered for re-use and the extract separated completely from the solvent. This is what makes this method better as there remains no trace of solvent on the extracts. At lower temperatures, it gives high yields of thermal labile compounds like nonflammable. It is however an expensive set up.

2.3.4 Sonication

In this sonochemistry based method, ultrasound waves (20 to 2000 KHz) are used to penetrate the sample materials. It can be used under normal temperatures but mostly on a hot plate at varied elevated temperature , thereby increasing cell wall permeability. Choice of solvents is critical based on the viscosity, polarity, surface tension and vapor pressure which influence the cavitation phenomena. Methanol, ethanol and hexane are the most common solvents, to which sometimes water is added. This method is effective in releasing components but its disadvantages include high installation and operational costs, modification of some active

Analytical techniques in the Separation and purification of plant constituents

Due to the variability of the nature of phenolic compounds (Polarity, chemical structure, glycosidic linkages and spectral characteristics) there is no single method that may be universally appropriate for the separation of all extracts, and should be carefully selected The identification and isolation of bioactive compounds from herbal extracts is the starting point for drug development for potentially new mechanisms against human diseases. To purify, samples are subjected to a range of solvents of varied polarities and then separated using chromatographic techniques. There are two broad types of chromatography; liquid and gas chromatography. Liquid chromatographic techniques are techniques where the mobile phase is a liquid whilst gas chromatography has gas phase as the mobile phase. The mechanisms of interaction vary depending on the states of the stationery phase and type of equilibrium reached. This section discusses basic mechanisms of both types