Mountain View High School, Mountain View, CA
Natural remedies for oral care have been used since ancient times. The fruit of Terminalia Chebula (T. chebula), a tropical deciduous tree, has been widely used in oral care in tropical countries. In more recent studies the antimicrobial and antibacterial properties of T. chebula have been adequately demonstrated. T. chebula, however, grows only in specific locations in South Asia and not easily available in other parts of the world. This paper explores the efficacy of the bark of the White Oak, Quercus Alba, a very common tree in North America as a low cost and widely available alternative to T. chebula in oral care. This study involved extraction of T. chebula and Q. alba furthermore method development by Thin Layer Chromatography (TLC). For the first time, different compounds were extracted, optimized and characterized by TLC and compared with that of T. chebula. Results from Thin Layer Chromatography run under identical conditions from the two species were similar. This indicated that the key constituents of Q. alba were highly likely to be the same as that of T. chebula warranting further confirmation through other analytical techniques. This work is paving the way for further characterization of Q. alba’s antimicrobial and antibacterial properties with the vision of developing natural oral care products.
Oral healthcare plays an important role in our daily life. Major problems can stem from unhealthy oral hygiene, such as gingivitis, and halitosis1. While there are plenty of oral care products in the market, most are artificially produced and are comprised of artificial/non-natural chemicals. The side effects of prolonged use of such formulations are largely unknown. Recently, oral hygiene products based on natural formulations have been getting a lot of attention.
For thousands of years, ancient people have utilized herbs and other natural remedies to treat a variety of medical conditions from back pain to oral care. The risk of side effects of these natural medicines are generally lower than artificial chemicals and that in turn has resulted in increased interests and associated funding for herbal remedy research.8 Recently, scientists have studied the pharmacology behind some of these ancient solutions and started creating formulations based on these natural medicines or medications. Simple techniques such cold water and hot water extraction to isolate key components have been developed and are widely used. Examples of these techniques include infusion (“soaking” in solvent such as water), sonication, and consequent filtration.
A key example of a natural oral health remedy is the fruit of T. chebula. T. chebula, a tropical species, has been used for thousands of years (since Vedic times in ancient India). Dried and powdered fruit of T. chebula was mixed with warm water and used as an oral rinse. T. chebula’s natural antimicrobial and antibacterial properties especially with regard to oral care have been proven.
This work explores the development of a natural oral care product based on the bark of the White Oak (Q. alba) tree as a low cost and widely available alternative to T. chebula. The work involved the extraction of T. chebula and Q. alba as well as the optimization of their characterization through Thin Layer Chromatography and comparative studies.
3.1 T. chebula
Figure 1: T. chebula Fruit- T. Chebula’s fruit is the source of its antimicrobial function. The fruit contains gallic acid and tannic acid, which are key in antimicrobial use.6
- chebula is a deciduous tree native to Southeast Asia, specifically in India, Nepal, and Southwest China. It is commonly referred to as Haritaki in the Indian subcontinent. T. chebula has ovoid leaves and these species can grow to 100 ft. The fruits are drupes, ranging in color from yellow to orange-brown.2 Figure 1 shows the T. chebula fruit.
3.1.2 Chemical Composition
While Tannic acid is the main constituent of T. chebula, it also contains phenolic compounds such as ellagic acid, 2,4-chebulyl-?-D-glucopyranose, chebulinic acid, gallic acid, ethyl gallate and punicalagin.2
3.1.3 Medicinal Uses
Traditionally, T. chebula has been used for the treatment of various medical conditions including acid reflux and gastro intestinal disorders.2 However, the main topical use of T. chebula is as a mouthwash due to its antimicrobial and antibacterial properties. It is also effective in the treatment of aphthous ulcer, as well as other oral diseases, such as gingivitis.2
3.2 White Oak (Q. alba)
Figure 2: Q. alba Tree5
White Oak (Q. alba) is a hardwood tree with light grey bark. This species is widely found in eastern and central North America.3 Figure 2 shows the Q. alba tree.
3.2.2 Chemical Composition
The bark of Q. alba contains phenolic compounds such as Grandinin/roburin E, castalagin/vescalagin, gallic acid, monogalloyl glucose, and valoneic acid dilactone, monogalloyl glucose, digalloyl glucose, trigalloyl glucose, ellagic acid rhamnose, quercitrin and ellagic acid.3 Some of the constituents of Q. alba such as ellagic acid, have been proven to have antimicrobial properties, antiproliferative and antioxidant properties are same as that of T. chebula. This was one of the main targets of this work.
4.0 Thin Layer Chromatography (TLC)
TLC was the main analytical technique used in this study. TLC is a simple, quick, and inexpensive procedure to identify the components of a particular mixture. For TLC, as in all forms of chromatography, a stationary phase (TLC plate) and a mobile phase (solvent system) are present. The mobile phase moves through the stationary phase, carrying the components of the mixture. Materials separate from each other based on the interaction with the solid phase. The sample to be analyzed is spotted onto a TLC plate (a sheet of aluminum or glass coated with an absorptive silica layer, otherwise known as stationary phase). Spotting is performed by dropping part of the mixture to be analyzed onto the TLC plate with use of a micro-capillary. The TLC plate is then kept in a solvent chamber with only the bottom of the plate dipped in the solvent solution.
4.1 Capillary Action
Capillary action causes the solvent to rise through the plate carrying with it the various constituents of the mixture. Capillary action is defined as the ability of a liquid to flow through narrow spaces despite gravity via intermolecular forces between the liquid and solid surroundings. Capillary action is the main driving force behind TLC; specifically, within the stationary phase. For the TLC techniques used in this work, silica (SiO2) was the adsorbent used as the stationary phase. The structure of silica consists of an oxygen and silicon atom bond to each other. At the surface of every particle residual, uncondensed hydroxyl groups from the original polymeric silicic acid remain, granting the silica gel its polymeric properties. These groups form bonded phases by reacting with the reagents of silane.11 Due to the complexity of the silica surface, it holds more than one hydroxyl group type; specifically, see figure 2 below. Different constituents move through varying distances, due to differences in their attraction to the TLC plate and solubility in the solvent system. This allows for the separation and analysis of the different materials.
Figure 2: Silica Structure - Silicon’s bond with oxygen through siloxane bonds.11
4.2 Rf Values
Rf value of a given component in the mixture is defined as the ratio of the distance it travels to the distance travelled by the solvent. The Rf value is dependent upon a number of parameters including the solvent, adsorbent, thickness of the adsorbent, amount of material spotted, temperature and the chemical properties of the components. Given these conditions can vary from experiment to experiment, relative Rf values are used when comparing two compounds. By comparing the Rf of an unknown compound to a known compound (preferably both run on the same TLC plate), the compound can be well defined. When the solvent has reached the desired point (the maximum point that the solvent can travel up the plate, indicated by how far the wetness rises through the plate), the plate is dried, and analyzed under a UV Lamp to identify separation of constituents. Figure 3 shows the experimental setup of a Thin Layer Chromatography system.
Figure 3: TLC Chamber and Plate - The plate is the square “paper” inside of the chamber containing the solvent system. The spot on the paper is the analyte.
5.0 Procedure and Analysis
Multiple extraction procedures were utilized in order to use each species for further studies.
5.1 Extraction and Analysis of T. chebula
5.1.1 Solid Extraction
The first step in preparation of the extract was to isolate T. chebula to its purest form. Figure 4 shows the “purified” dried T. chebula. These fruits were obtained from the Asthagiri Herbal Research Foundation, Chennai, India. The ‘pure’ product was ground to a fine powder using a commercially available mixer (Preethi). 200 g of the product was sieved and resultant material was measured using a weighing scale (Figure 5) to be 152 g.
Figure 5: Weighing Scale
5.1.2 Initial Aqueous Extraction For TLC Purposes
To make the first aqueous extract of T. chebula, a simple water extraction procedure was utilized by adding 200 mL of water to 5 g of the ground fruit. It was then sonicated for 5 minutes. Sonication is the act of applying sound energy to agitate particles in a sample, for various purposes.4 Figure 6 shows the sonicator used in the lab. This equipment is a device that emits vibration patterns through the medium of water to agitate the mixture. All test tubes and flasks with standard thickness can be used. The T. chebula sonicated mixture was placed in a standard borosilicate test tube with 9 mL volume, and was covered with plastic wrap to prevent spillage.
Figure 7: Initial Aqueous Extract after Sonication
In order to further purify the extract, and possibly allow for better separation the sonicated extract was filtered twice using filter paper and separation funnel. Figure 7 and 8 show the initial and final filtered extracts.
Figure 8: Final Filtered Aqueous Extract of T. chebula
5.1.4 Mobile Phase Optimization
The solvent system is called the mobile phase in TLC. This solvent system (constituents and their ratio) has to be optimized for a particular TLC system and this optimization is done experimentally. The plate used in this TLC was 1.5 x 2 x 7 cm. After a rough extraction of T. chebula, experiments were undertaken to find this ideal solvent system. Given there was limited prior work in solvent mixture studies for T. chebula7, additional mobile phases were experimented with in order to arrive at an optimum one. These are listed in table 1. Five trials were done for each mobile phase. Representative plates photographed under Ultraviolet light (UV) is shown in the Figure 9. Figure 9a shows an example of a TLC plate with a mobile phase (Toluene: Ethyl Acetate: Formic Acid: Methanol (6:6:1.8:0.25) that has poor clarity and weak separation. Figure 9 b shows the TLC plate for Ethyl Acetate: Toluene: Formic Acid: Acetic Acid: Water (6:3:0.5:0.5) system. While it shows general separation, the spot clarity is not as good as figure 9c, that illustrates the most optimal system – Ethyl Acetate: Formic Acid: Acetic Acid: Water (10:1.1:1.1:0.6) for TLC characterization of T. chebula.
|Mobile Phase Constituents||Ratio (mL)|
|1||Toluene: Ethyl Acetate: Formic Acid: Methanol||(6:6:1.8:0.25)|
|2||Toluene: Acetone: Glacial Acetic Acid||(3:1:2)|
|3||Toluene: Ethyl Acetate: Formic Acid: Methanol||(4.3:4.3:1.2:0.3)|
|4||Toluene Ethyl Acetate: Formic Acid||(5:4:1)|
|5||Ethanol: Glacial Acetic Acid: Toluene||(5.5:1:1.5)|
|6||Ethyl Acetate: Formic Acid: Acetic Acid: Water||(10:1.1:1.1:0.6)|
|7||Ethyl Acetate: Toluene: Formic Acid: Acetic Acid: Water||(8:2:1.1:1.1:0.6)|
|8||Ethyl Acetate: Toluene: Formic Acid: Water||(6:3:0.5:0.5)|
Table 1: Mobile Phases Used for T. chebula
Figure 9: TLC Plate for representative mobile systems
(a) Ethyl Acetate: Formic Acid: Methanol (6:6:1.8:0.25)- Example of poor solvent system, unable to determine results
(b) Ethyl Acetate: Toluene: Formic Acid: Acetic Acid: Water (6:3:0.5:0.5)- Example of clearer spot formation, still not clear enough for determinative results
(c) Ethyl Acetate: Formic Acid: Acetic Acid: Water (10:1.1:1.1:0.6)- Optimized TLC system, clear spot formation
5.2 Extraction and Analysis of Q. alba
The bark of Q. alba was pulverized using a commercially available mixer (Preethi) and water was added to it. Multiple extraction techniques were explored to optimize the extraction process. These are detailed in the section below.
5.2.1 Sonicated Extract Procedure
5 grams of Q. alba bark was measured and placed in two different tubes by using the weighing scale (4.9002 g, 5.0055 g). 20 mL of water was added to both extracts and then sonicated for 5 and 10 min respectively. Figure 10 represents these samples.
Figure 10: Sonicated for 5 min and 10 min Q. alba Aqueous Extracts
5.2.2 Microwaved Extract Procedure
Figure 11: Microwave Q. alba Extract
5.2.3 TLC Procedure: Mobile Phase Optimization
There was no prior work or literature in mobile phase optimization of Q. alba extracts. The various mobile systems utilized in this study are shown in Table 2. Three different extracts (5 min sonication, 10 min sonication and microwave) were run on TLC with multiple mobile phases shown in table 2. The microwave extract samples did not separate. The 5 and 10 min sonication did show separation, although after 5 min the separation seemed to be a better method. Figure 12 shows photographs of the TLC plate under ultraviolet light. The top three lines represents Ethyl Acetate: Formic Acid: Acetic Acid: Water (10:1.1:1.1:0.6) as the mobile phase with various samples. The bottom three lines represent Toluene: Ethyl Acetate: Hexane: Methanol: Water: Formic Acid (3:3:2:1:0.5:0.5) as the mobile phase. The Ethyl acetate: Formic Acid: Acetic Acid: Water (10:1.1:1.1:0.6) was found to be the optimal mobile phase for TLC of Q. alba.
|1||Methanol: Water: Acetic Acid||(0.1:8.9:1)|
|2||Ethyl Acetate: Formic Acid: Acetic Acid: Water||(10:1.1:1.1:0.6)|
|3||Toluene: Ethyl Acetate: Hexane: Methanol: Water: Formic Acid||(3:3:2:1:0.5:0.5)|
Figure12: TLC Plates for representative mobile systems
(1) Ethyl Acetate: Formic Acid: Acetic Acid: Water (10:1.1:1.1:0.6); (a) 10 min sonication, (b) 5 min sonication, and (c) Microwave
(2) Toluene: Ethyl Acetate: Hexane: Methanol: Water: Formic Acid (3:3:2:1:0.5:0.5); (a) 10 min sonication, (b) 5 min sonication, and (c) Microwave
The process for the TLC characterization of T. chebula and Q. alba was studied and optimized. From Figures 9 and 12, one found that the separated constituents in both the TLCs were at the same location on the plate. The solvent front was also at the same location in both cases indicating the Rf values could be similar. Rf values, the ratios between the amount of distance the constituent traveled to the distance the solvent front traveled were calculated to be 0.64 and 0.65 for T. chebula and Q. alba, respectively. In most cases, if the Rf values were different that would represent a clear confirmation of different compounds7. However, while similar Rf values based on tests run under identical conditions would strongly indicate similar constituents, additional confirmatory tests would be required.9 In this work it was found that the Rf values of T. chebula and Q. alba were almost the same, strongly indicating the presence of similar compounds7, warranting further confirmation through analytical techniques such as FT-IR, Raman, NMR or Mass Spectroscopy.
Possible sources of error in the work include inaccuracy in human spotting on the stationary phase/TLC plate, as well as in U.V. analysis/measurement. Machine spotting was identified to be one key improvement area.
In this study, for the first time, the mobile phases of Q. alba were extracted and characterized through Thin Layer Chromatography as well as compared with T. chebula. The TLC results from the two species were very similar strongly indicating that they may have similar compounds. Results from this work demands further confirmation with other spectroscopic methods as well as characterization of Q. alba’s anti-bacterial and anti-microbial properties. Further research would involve utilizing a comparative microbial lab study of T. chebula and Q. alba in order to support our claims as well as pave the way towards the development of a new natural oral care product based on Q. alba.
The author would like to acknowledge Dr. Narasimhan, CEO and Founder of Asthagiri Herbal Research Foundation (India) for gracious support of this work and the research team at Asthagiri for technical support and guidance.
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