An Integral Component of Indian Ayurvedic Medicine System - Medical Science Assignment Help

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INVITRO CULTURE STUDIES OF BACOPA MONNIERI
ABSTRACT
Brahmi (Bacopa monnieri), an integral component of Indian Ayurvedic medicine system, is facing a threat of extinction owing to the depletion of its natural populations.Enhanced production of secondary metabolites was obtained from suspension cultures of Bacopa monnieri.our studies can be utilised in meeting the demand and supply ratio of the drug with constant and elevated production of secondary metabolites produced in short duration. the growth and content of bacoside-A, an important pharmaceutical compound in B. monnieri, were investigated under secondary metabolites by elicitation process .
Introduction
Bacopa monnieri is a medicinal plant commonly termed Brahmi, herb of grace, thyme-leaved Graciela, and water hyssop (Raman, 2019). It grows in wet and humid habitats, generally in tropical environments. The hiding properties of Brahmi include; its high enrichment with antioxidants, inflammation-reducing property, boosting brain functions, help in managing attention deficit hyperactivity disorder (ADHD), and many more (Raman, 2019). With its continuous exploitation for its medicinal benefits, Brahmi is on verge of being extinct. Using Vegetative propagation or micropropagation techniques Bacopa monnieri can be propagated in in-vitro conditions with suitable conditions needed (Karata? et al., 2016). Micropropagation of Bacopa monnieri can be done by following steps:
 

Explant selection for callus induction
Bacopa monnieri can be propagated in lab conditions that require certain materials like disease and contamination-free explant, laminar airflow to provide sterile conditions, and some tools and chemicals (Behera, et al., 2015). The nodal segment, shoot tips, or a leaf can be selected as explant. The explant is first washed under running water for around 20-30 minutes. Then add a few drops (5-6) of lavolene (a mild detergent) for 10-12 minutes. After this, soak the explant in 0.2% (w/v) Bavistin (fungicide) for 10 minutes. Followed by repeated washing with distilled water and a final treatment with 0.1% (w/v) HgCl2 for 5 minutes. The final steps would be done under laminar airflow to avoid any chances of contamination. After HgCl2 treatment wash the sample with distilled water around five to six times to remove traces of HgCl2 which can interfere in further steps. In the end wash the explant with 70% ethanol (disinfectant) for 1 minute followed by washing with distilled water (2 to 3 times) (Sharma, & Khan,2011).
 

Culture media preparation for enhanced bacosides production
Bacosides are chemical compounds produced by Bacopa monnieri(BM) which are used for medicinal purposes (Sekhar, Viswanathan, & Baby, 2019). A suspension basal media (HI Foliar media) can be prepared by mixing 3g media in one liter of buffer (Chaturvedi, &Hingorani, 2018). Other compositions of the media include tyrosine and phenylalanine precursors. Copper sulfate, cobalt chloride, and zinc chloride were added to added as stress-producing compounds. Chitinphilus and Streptomyces both act biotic stress inducers against BM and lead to reduced gelling index, reproduction factor and elevate the production of secondary metabolites like bacosides (Gupta, et al., 2017). Then explant was added to the media in completely sterile conditions followed by covering the open area of the plate. After this, the plantlets were kept as completely closed with the help of parafilm to avoid entry of contaminates. For proper growth of the plant at a set cycle of light-dark needed to be given which was set as 16 hours light and 8 hours of the dark period. The pH value of the media was set as 5.8 and growth was monitored at room temperature for around one to two weeks (Chaturvedi, &Hingorani, 2018).
 

Enhanced production of secondary metabolites using elicitors
Elicitors are of two types; biotic and abiotic which act as the inducers to trigger the production of secondary metabolites by the plant by creating stressed conditions. They may be inducing the production endogenously as well as exogenously (Narayani & Srivastava, 2017). They also act by rearranging the metabolic fluxes between an uncontrolled expressed pathway and an elicitor-induced pathway. Polysaccharides from plant cell walls and microorganisms act as biotic elicitors whereas UV radiation, salts of heavy metal, and chemicals act as abiotic elicitors. Biotic elicitors are thought to act by binding themselves to specific protein receptors present on the surface followed by inactivation of ATPase embedded in the cell membrane. It leads to proton electrochemical gradient imbalance across the membrane (Katare, et al., 2009). The scenario is reversed in the case of abiotic elicitors which generally target the intracellular compartment of the cell. Chitinphilus and Streptomyces are the biotic elicitors to induce enhanced production of bacosides from BM (Gupta, et al., 2017). Whereas, Copper sulfate, cobalt chloride, and zinc chloride are the abiotic stressors (Chaturvedi &Hingorani, 2018). To Enhance the Secondary metabolites production by elicitation

Plant secondary metabolites are playing important role in relieve the several nourishments in the traditional medicine and peoples uses. Plant secondary metabolites are classified into many classes based on the chemical structure (Santos, et al., 2016). Plant secondary metabolites are having many applications in human health and nutritional aspect. Productions of secondary metabolites are commercial importance in medicine, food additives, pharmaceuticals and industrial materials (Kharde, et al., 2018). Different types of phenols were found in the plant leaves by using the elicitations (different types of microbial treatment) to influence the secondary metabolites (Santos, et al., 2016).

To influence the production of secondary metabolites by using elicitation in the Bacopa monnieri plant: Elicitation is the one of the most popular techniques nowadays used for improving the biotechnological production of secondary metabolites. These studies indicate the effectiveness of biotic and abiotic elicitations on Bacoside production in Invitro culture of this medicinal properties of Bacopa monnieri it is important herb known for its memory enhancer. In this paper were studies of influence the production of secondary metabolite content (Mustafa, et al., 2011).
To enhancement of secondary metabolite as well as biomass accumulation under Invitro conditions such as optimization of the medium and culture environment, selection of high secondary metabolite producing cell lines, use of organic supplements, elicitors and precursors in the medium. To influence the production of secondary metabolites by different types of abiotic and biotic factors are called elicitors. Elicitation is complicated process. It is depending on the many factors such as elicitation concentration, explants growth stage of the culture at the time of elicitor addition(accession) and exposure time with elicitor. Plant growth regulators also used in the abiotic elicitors then played in the important role in the enhancement of secondary metabolites (Bacoside) content in) Invitro culture conditions. The stress, including various elicitors or signal molecules, often induces the secondary metabolite production in the plant tissue culture system (Kharde, et al., 2018).
To determining the chemical composition and therapeutic activity of medicinal plants there is stress is the important factor. Elicitation is the stress response to induce the desired chemical response to the plants. Elicitation can define as a substance for stress factor. Elicitation was applied in very low quantity to living system and it is improving the biosynthesis of desired compound. Elicitation is the enhancing the secondary metabolites production because addition of the trace number (amount) of elicitors. Elicitation is recognized the practically feasible strategy for enhance the production of secondary metabolites from cell, organ and plant system. Elicitors are the chemical compounds from abiotic and biotic. That can excite stress responses in plants, leading to the enhanced synthesis and collection of secondary metabolites or the induction of novel secondary metabolites (Kharde, et al., 2018).

Elicitation is classified into two types based on the nature. There are 1. abiotic elicitation 2. biotic elicitations. The abiotic elicitations are form of substance that is non biological origin. The abiotic elicitation is classified into 3 types they are physical, chemical and hormonal factor. In the chemical factors are heavy metals, mineral salts and gaseous toxins comes under this. Physical factors are UV radiation, thermal stress, osmotic stress, salinity and Drought are coming under this category. A different type of plant growth hormones comes under the hormonal factors. The biotic elicitations are form of substance that is biological origins. The biotic elicitation classified into 4 types there are polysaccharides, yeast extract, fungi and microorganisms (Kharde, et al., 2018).
4.1.2 Abiotic elicitation:
Abiotic elicitation classified into 3 types, there are physical, chemical and hormonal Physical stress Salts stress:
Salts are one of the most using the elicitation to production of the secondary metabolites. when the plants are given salinity conditions, they accumulate secondary compounds like phenol, terpene and alkaloid. The salt stress elicitation was reported that Bacopa monnieri plants cultures produced Bacoside A content in large amounts when given salts like potassium chloride and calcium chloride treatments (Showkat, et al., 2010, Parale, et al., 2010).
Drought stress: -
The growth in plants as well as their reproductive development drought is very important factor. All plant species possess different tolerance from drought stress. A study showed that plants were provides the drought conditions then the plants are release the greater number of secondary metabolites (Showkat, et al., 2010,Sugimoto, et al., 2010).
Thermal stress:
Thermal stress is the extreme temperature conditions like elevated temperature (hot stress) and low temperature (cold stress) can be restrict the growth of the plants. The thermal stress also influences the secondary metabolite production. In normally to induce callus tissues a temperature range 17-25 degree Celsius is used (Showkat, et al., 2010).
Osmotic stress:
Osmotic stress is the one of the important physical elicitors by using to enhance the secondary metabolite production. Osmotic stress is known as water stress. Osmotic stress can alter the physiological and biochemical properties and increase the concentration of the amount of the secondary metabolites content in the plants (Kharde, et al., 2018,Parale, et al., 2010).
4.1.3Classification of elicitors (based on the nature)



Chemicals elicitors: -
Chemical elicitors are major role in the Enhance the production of the secondary metabolites; chemical elicitors are heavy metals, mineral salts and gaseous toxins. It is thought to be an abiotic stress agent for living organisms because of them to increase use in the developing fields of industries, agrotechnology and high bioaccumulation and toxicity. A study reported that metals such as Ni, Ag, and Fe, and co have been shown to elicit the enhancement the production of secondary metabolites in many plants (Sharma, et al., 2015, Lal, et al., 2017).
In the review paper additionally using silver nitrate nanoparticles, the silver nano particles are being extensively using many biological sources they are including fungi, bacteria and plants. Silver nano particles are also applied to elicitation mechanism to enhance the production of secondary metabolites in the Invitro culture. The silver nanoparticles are synthesis is getting more popular because the silver nanoparticles are high reactivity of plant extract and easily available of plant, materials (Lal, et al., 2017).
Hormonal elicitors: -
Hormonal elicitors have also been using the enhance the production of secondary metabolites using various hormones. The hormonal elicitation was most studies because they are key role in the defense mechanisms in the plants (Parale, et al., 2010).
Biotic elicitation: -
Biotic elicitation was polysaccharides, fungi, bacterial, and yeast extract extra.
Polysaccharides:
Polysaccharides are major role in the production of secondary metabolites. In the polysaccharides are proteins, carbohydrates and hormones their adding the polysaccharides in to the cultures exert the stress conditions then release the secondary metabolites. (Naik, et al., 2016)
Fungal: -
In the plant tissue culture to enhance the secondary metabolite production to using the biotic elicitation by fungal origin was widely using. This elicitation has been effective in stimulating the production of secondary metabolites. It is well known as specific chemical interaction between the microorganism and higher plants exert stress into the plants to influence the growth and development. To overcome this stress conditions plats are production of bioactive compounds. Aspergillus Niger was common soil born pathogenic fungus. Aspergillus Niger was widely used as elicitors for enhancement of secondary metabolites in Invitro conditions (Varghese, et al.,2020).
Bacterial: -
The bacterial elicitors stimulated the biosynthesis of saponins in adventitious hairy root cultures. Agrobacterium rhizogenes, bacillus species are using this type of elicitation in the Bacopa monnieri.
Agrobacterium rhizogenes can cause the hairy roots then the plants can feels stress conditions they can enhance the production of secondary metabolite content. (Kharde, et al., 2018)
HPLC Analysis of in vivo and in vitro produced bacosides
For the analysis of bacosides produced by BM, include a series of set protocols. The first step was to harvest BM plant materials and treat them thoroughly. The materials were then cut into minute pieces and the extracts were obtained using the methanolic cold extraction method. After this, extracted samples were kept in methanol for two days followed by filtration and drying in a vacuum (Chaturvedi, &Hingorani, 2018). These dried samples were then prepared for high-performance liquid chromatography (HPLC) which is a quantitative as well as a qualitative assay and helps in detecting the number of bacosides with their level of purity (Jauhari et al., 2019; Indian Institute of Technology Kanpur, 2012). HPLC was performed using a shim-pack GIST C18 packed column. Eluents will be detected at a wavelength of 205 nanometers (Chaturvedi, & Hingorani, 2018).
To develop a highly reproducible protocol for plant regeneration via organogenesis from various explants of Bacopa monnieri
Organogenesis is a process by which a callus from explant is differentiated into plant organs like roots, shoots, and buds. For organogenesis of BM explants, the callus needs to be incubated in MS media with different combinations and concentrations of plant growth hormones for example NAA, 2, 4-D, and kinetin. After the inoculation of the culture with the explant, the culture containing plates needs to be completely sealed. Label the plates properly and then place them for incubation at room temperature in a rack with a regulated supply of light (Samanta, Mallick, & Roy, 2019).
Protocol for in vitro rooting and acclimatization
After 10-15 days of explant inoculation into the media, shoots were developed. The newly born micro shoots then sub-cultured into fresh media after their proper growth for 2-3 weeks. Shoot induction is done first to make the plant capable of photosynthesizing. Now, micro shoots need to be transferred into rooting media for root development. The generated shoots were then chopped down using forceps under the laminar hood and the rest of the plant part was then placed on rooting media with (0.1-1.0 mg L-1) of indole-3-butyric acid (IBA) and left for incubation in the growth room (Behera, et al., 2015). In the acclimatization process, the rooted plants were taken out carefully from the media and washed properly under running water to remove the agar. After that, the plants were then transferred to plastic bags filled with sterile soil. The platelets needed to be covered with plastic to maintain humidity conditions (90 percent) for proper growth. Irrigation was done after 2 days using MS salt solution (half strength and without vitamins) for two weeks. Plastic covers then removed after completion of 2 weeks. After this, the plants with new leaves were then transferred to pots filled with soil and vermin-compost in equal proportion. The plants were placed in a greenhouse for 2 weeks and then finally brought to direct sunlight(Samanta, Mallick, & Roy, 2019) (Sharma, & Khan, 2011).
Conclusion
Bacopa monnieri plants were successfully grown after following the right protocols taking from explant isolation to acclimatization. The plants generated with the technique were healthy and diseased-free and were quite adapted to the conditions. Both, biotic and abiotic factors were used to induce sufficient bacoside production. The quality and quantity of bacosides were successfully achieved using HPLC. The errors reported in the process were minimum. All the precautions related to the experiment were taken into consideration.
 

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